A γ-lactam siderophore antibiotic effective against multidrug-resistant Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter spp.

Article abstract of DOI:10.1016/j.ejmech.2021.113436

Serious infections caused by multidrug-resistant (MDR) organisms (Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii) present a critical need for innovative drug development. Herein, we describe the preclinical evaluation of YU253911,

Full text of DOI:10.1016/j.ejmech.2021.113436

European Journal of Medicinal Chemistry 220 (2021) 113436
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
A
g
-lactam siderophore antibiotic effective against multidrug-
resistant Pseudomonas aeruginosa, Klebsiella pneumoniae, and
Acinetobacter spp.
a
b
c
c
Joel A. Goldberg , Vijay Kumar , Elizabeth J. Spencer , Denton Hoyer ,
a
a,
d
a,
d
a
Steven H. Marshall , Andrea M. Hujer , Kristine M. Hujer , Christopher R. Bethel ,
a,
b,
d
a,
d,
e
d, f
Krisztina M. Papp-Wallace
, Federico Perez
, Michael R. Jacobs
,
g
h
b,
**
, Mark S. Plummer c
, ***
David van Duin , Barry N. Kreiswirth , Focco van den Akker
,
a, b, d, e, i, j, *
Robert A. Bonomo
Research Service, Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Cleveland, OH, 44106, USA
Department of Biochemistry, Case Western Reserve University, Cleveland, OH, 44106, USA
Yale Center for Molecular Discovery, West Haven, CT, 06516, USA
a
b
c
d
e
f
Department of Medicine, Case Western Reserve University, Cleveland, OH, 44106, USA
Geriatric Research, Education and Clinical Center, Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Cleveland, OH, 44106, USA
Department of Pathology, University Hospitals Cleveland Medical Center, Division of Clinical Microbiology, Cleveland, OH, 44106, USA
University of North Carolina School of Medicine, Chapel Hill, NC, 27514, USA
Center for Discovery and Innovation, Hackensack Meridian Health, Nutley, NJ, 07601, USA
Departments of Pharmacology, Molecular Biology & Microbiology, And Proteomics & Bioinformatics, Case Western Reserve University, Cleveland, OH,
g
h
i
4
4106, USA
j
CWRU-Cleveland VAMC Center for Antimicrobial Resistance and Epidemiology (Case VA CARES), Cleveland, OH, 44106, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 February 2021
Received in revised form
Serious infections caused by multidrug-resistant (MDR) organisms (Klebsiella pneumoniae, Pseudomonas
aeruginosa, Acinetobacter baumannii) present a critical need for innovative drug development. Herein, we
describe the preclinical evaluation of YU253911, 2, a novel g-lactam siderophore antibiotic with potent
antimicrobial activity against MDR Gram-negative pathogens. Penicillin-binding protein (PBP) 3 was
shown to be a target of 2 using a binding assay with purified P. aeruginosa PBP3. The specific binding
interactions with P. aeruginosa were further characterized with a high-resolution (2.0 Å) X-ray structure
of the compound’s acylation product in P. aeruginosa PBP3. Compound 2 was shown to have a concen-
23 March 2021
Accepted 30 March 2021
Available online 8 April 2021
Keywords:
tration >1
Employing a meropenem resistant strain of P. aeruginosa, 2 was shown to have dose-dependent efficacy
at 50 and 100 mg/kg q6h dosing in a mouse thigh infection model. Lastly, we showed that a novel
lactam and -lactamase inhibitor (BLI) combination can effectively lower minimum inhibitory concen-
mg/ml at the 6 h time point when administered intravenously or subcutaneously in mice.
g
-Lactam
Siderophore
Multidrug-resistant gram-negative
pathogens
g-
b
Penicillin-binding protein
trations (MICs) against carbapenem resistant Acinetobacter spp. that demonstrated decreased suscepti-
bility to 2 alone.
©
2021 Published by Elsevier Masson SAS.
Abbreviations: MDR, multidrug-resistant; PBP, penicillin-binding protein; MIC, minimal inhibitory concentration; blas,
muscularly; CFU, colony-forming unit; WGS, whole-genome sequencing; CRAB, carbapenem-resistant Acinetobacter baumannii; CSAB, carbapenem-susceptible Acinetobacter
baumannii; BLIs, -lactamase inhibitors; LCMS, liquid chromatography mass spectrometry; NMR, nuclear magnetic resonance; UV, ultraviolet; DMSO, dimethylsulfoxide;
MSTFA, N-methyl-N-(trimethylsilyl)-trifluoroacetamide; AN, Acinetobacter nosocomialis; FI, fluorescence intensity; SAR, structure activity relationship.
Corresponding author. Research Service, Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Cleveland, OH, 44106, USA.
b-lactamases; SC, subcutaneously; IM, intra-
b
*
*
*
* Corresponding author.
** Corresponding author.
E-mail addresses: fxv5@case.edu (F. van den Akker), marksplummer@gmail.com (M.S. Plummer), Robert.Bonomo@va.gov (R.A. Bonomo).
https://doi.org/10.1016/j.ejmech.2021.113436
223-5234/© 2021 Published by Elsevier Masson SAS.
0

J.A. Goldberg, V. Kumar, E.J. Spencer et al.
European Journal of Medicinal Chemistry 220 (2021) 113436
1
. Introduction
bacteria [22]. In an effort to explore this finding, additional ana-
logs with modified side chains were screened, leading to the
identification of 2 which contains a chloroaminothiazole group.
This group was found to favorably modulate several biological
properties (vide infra). The synthesis of 2 parallels the synthesis of 1
and employs a common advanced intermediate dihydroxyph-
thalimide [26] -appended bicyclic pyrazolidinone, 14 (details of the
synthesis of 14 are in the Supporting Information, pages 4e17).
Coupling of 14 using the appropriately protected chlor-
oaminothiazole 15 followed by deprotection and reverse phase
chromatography provides compound 2, Scheme 1. The chiral purity
of 2 was not assessed after the synthesis was completed though
Multidrug-resistant (MDR) bacterial infections pose an
increasing threat to public health, causing significant morbidity,
mortality, and economic hardship. In the United States alone, more
than three million infections each year are caused by antibiotic-
resistant bacteria resulting in approximately 36,000 deaths and
more than $20 billion in healthcare costs [1]. The future global
impact is even more staggering with an estimated cumulative
economic cost of $100 trillion from now until 2050, with an asso-
ciated 10 million deaths, resulting from MDR infections [2]. Invasive
infections with Gram-negative bacterial pathogens in particular
have become increasingly problematic and are associated with 28
day mortality rates of 30e70% (https://www.cdc.gov/
drugresistance/biggest-threats.html). Resistant strains of Acineto-
bacter baumannii, Pseudomonas aeruginosa, and carbapenem-
resistant Enterobacterales are identified as “urgent” and “serious”
threats by the CDC and WHO, illustrating the critical need for new
therapeutics [3e7].
The “hard-to-treat” nature of these infections is often caused by
extensive antibiotic-resistant phenotypes that allow pathogens to
overcome standard antibiotic regimens [8]. Despite the increasing
public health threat, few truly novel agents are in development to
treat such infections, as most large pharmaceutical companies have
withdrawn from antibacterial research [3,4,9,10]. Government
agencies are aware of the ever-growing issue of antibacterial
resistance and are responding with several initiatives intended to
incentivize renewed efforts in antibiotic development [11]. They
include public-private partnerships highlighting the urgent need
for novel treatments.
Boc-
2.2. Microbiology
2.2.1. -Lactam 2 inhibits the growth of MDR Gram-negative bacilli
L-serine was employed as the starting material.
g
A comparison of minimum inhibitory concentrations (MICs) for
2 and 1 vs. previously described clinical carbapenem-resistant
isolates of P. aeruginosa [27] and K. pneumoniae [28] are provided
in Fig. 2 [22]. MIC data for both 2 and 1 is provided for comparison;
the detailed values for each individual strain are in Supplemental
Table 1, page 18. In all but one case (YUKP-39), microbiologic po-
tency of 2 was maintained, although the activity trended to be
slightly less active for
K. pneumoniae. Nevertheless, MIC testing of 2 afforded an MIC50 of
0.5 and 1 g/mL against 23 samples each of MDR P. aeruginosa and
2 compared to 1, particularly for
m
K. pneumoniae, respectively. Notably, MIC data of 2 were signifi-
cantly lower than meropenem in all cases but 1 isolate (YUKP-39).
We purposely selected
a subset of our P. aeruginosa and
Resistance in multidrug-resistant Gram-negative organisms is
multifactorial. These causes may include reduced permeability,
modification of target proteins, and overexpression of efficient and
K. pneumoniae panels that had the most potent compound 1 MIC
values. Using such a narrow comparison means it is possible that 2
may not necessarily have poorer overall activity against larger
panels. Similar to what was previously reported for 1 [22], MIC
values were dependent on maintaining low iron concentrations in
the culture media (data not shown). All results were generated
using Chelex® resin-treated media to mimic the low iron concen-
trations found in vivo, and in accord with CLSI recommendations for
siderophore-containing antibiotics.
diverse efflux pumps [12,13]. For
concern is the increasing diversity of plasmid-mediated carbape-
nemases ( -lactamases (blas): NDM-1, KPC, and OXA-type class D
carbapenemases) making -lactam antibiotics ineffective, despite
their common coadministration with -lactamase inhibitors [1].
b-lactam antibiotics, a particular
b
b
b
We recently reported the initial attributes of an antibacterial
agent effective against Gram-negative pathogens based on a revi-
talized non-
b
-lactam pyrazolidinone scaffold [14e21] exemplified
2.2.2. The g-lactam 2 possesses enhanced antimicrobial activity
by 1 (Fig. 1) [22]. The strategy involves inhibiting penicillin-binding
against MDR A. baumannii
proteins (PBPs), which are validated targets for antibacterial dis-
MICs in low iron media against a 198-member panel of previ-
ously described Acinetobacter spp. clinical isolates, (79%
A. baumannii, 21% a mixture of A. nosocomialis, and A. pittii) [30] are
presented in Fig. 3 and Table 1. Activity against a 98-member
carbapenem-resistant A. baumannii subset of the panel is also
shown in Fig. 3 and Table 1. Importantly, the MIC values of 2
covery, while avoiding susceptibility to
b-lactamase inactivation.
This is accomplished by using a -lactam ring with tunable reac-
g
tivity as the molecule’s core [18,20]. Additionally, 1 includes a
siderophore moiety which exploits intrinsic bacterial iron transport
processes (“Trojan horse approach”) to overcome the decreased
permeability of Gram-negative bacteria [23e25]. Agent 1, which
compare favorably to all b-lactam classes, including aztreonam 3
contains a prototype
g
-lactam-siderophore, inactivates PBP3 and
(monobactam), ceftazidime 4 (cephalosporin) and meropenem 5
(carbapenem) (Table 1). Applying the breakpoints for cefiderocol 9
possesses excellent in vitro potency against MDR clinical isolates of
P. aeruginosa, K. pneumoniae, and E. coli. Herein, we report the
discovery and properties of YU253911, 2, a chloroaminothiazole
analog of 1 (Fig. 1), which possesses enhanced potency versus
Acinetobacter spp. and improved pharmacokinetics versus 1 as
illustrated by in vivo efficacy in a rodent thigh infection model
employing an MDR strain of P. aeruginosa.
(susceptibility ꢀ4
mg/mL and resistance ꢁ16
mg/mL), a similar
approved drug utilizing a siderophore transport mechanism [29], to
compound 2 results in a susceptibility to 2 of 86% (171 out of 198) of
the full Acinetobacter panel and 83% (81 out of 98) of the subset of
carbapenem-resistant A. baumannii (CRAB). Detailed values for
each individual strain are in Supplemental Table 2, page 19.
2
. Results and discussion
2.2.3. Combination of 2 with sulbactam further enhances growth
inhibition of Acinetobacter spp.
2.1. Chemistry
We investigated whether partner agents could improve the
already potent activity of 2 against resistant Acinetobacter spp.
We previously reported the synthesis and characterization of 1
growth.
tion with
hydrolysis. Although the
b
b
-lactam antibiotics are often used clinically in combina-
-lactamase inhibitors (BLIs) to protect against enzymatic
-lactam core of 2 possesses intrinsic
as a prototype of a new class of siderophore-conjugated
g-lactam
antibiotic with enhanced activity against MDR Gram-negative
g
2

J.A. Goldberg, V. Kumar, E.J. Spencer et al.
European Journal of Medicinal Chemistry 220 (2021) 113436
Fig. 1. Structures of
g-lactam siderophores 1 and 2 versus comparator b-lactam antibiotics and b-lactamase inhibitors. The b- or g-lactam core is highlighted in red; iron-binding
siderophore moieties in blue.
stability to
b
-lactamase hydrolysis (vide infra),
b
-lactamase in-
lactam 2 is believed to target PBP3 (vide infra) and inhibition of two
PBP enzymes would be expected to produce enhanced antimicro-
bial effects.
hibitors (BLIs) were nevertheless evaluated for their ability to
augment the breadth of 2 effectiveness against Acinetobacter spp.
Sulbactam 10, a BLI commonly marketed in combination with
ampicillin (12, Unasyn®), was selected for study as a potential
partner of 2 for two reasons. Firstly, sulbactam has been shown for
decades to be safe [31,32] and well-tolerated in patients [33], with
favorable pharmacokinetics [34,35]; secondly, sulbactam possesses
intrinsic antimicrobial activity against Acinetobacter spp. due to
postulated inhibition of PBP1 and PBP3 [36]. Furthermore, sulbac-
tam is currently under investigation in combination with durlo-
The 24 Acinetobacter isolates from Fig. 3 with higher MICs to
compound 2 (MICꢁ16
mg/mL) were tested versus sulbactam alone
or in combination with 2. MIC values show 79% (19/24) of the tested
isolates were found to have their growth inhibited to some extent
by sulbactam (MICsꢀ16
mg/mL) (Table 2). Furthermore, when 2 and
sulbactam were combined in a 1:1 ratio in a typical 2-fold MIC
dilution scheme, 50% (12/24) of the isolates previously resistant to
2 showed improved MICsꢀ4
mg/mL. Published human pharmaco-
logic data for ampicillin-sulbactam and sulbactam-durlobactam
bactam, 11, for the treatment of Acinetobacter infections. The
g-
3

J.A. Goldberg, V. Kumar, E.J. Spencer et al.
European Journal of Medicinal Chemistry 220 (2021) 113436
ꢂ
Scheme 1. Synthesis of YU253911, 2: (a) i. 15, Oxalyl chloride, catalytic DMF; ii) 14, MSTFA, Hunig’s base, 3.5 h, 100% crude. (b) 2:1 DCM/TFA, triethylsilane, 0 C to RT, 1.5 h, toluene
chase, reverse phase MPLC C18, 0e60% acetonitrile 0.1% formic acid/water with 0.1% formic acid, 30%.
Fig. 2. Compound 2 and 1 MIC values against representative g-lactam susceptible/carbapenem-resistant K. pneumoniae and P. aeruginosa strains (23 each). Except for one isolate,
YUKP-39, compound 2 maintains microbiologic potency against MDR Gram-negative rods previously described for this g-lactam-siderophore class (see also Supplemental Table 1,
page 18).
Fig. 3. Distribution of MICs in low iron media of 2 (
mg/mL) against a 198-member Acinetobacter spp. panel and subset of 98 carbapenem-resistant A. baumannii (CRAB) isolates. See
also Supplemental Table 2, page 19.
4

J.A. Goldberg, V. Kumar, E.J. Spencer et al.
European Journal of Medicinal Chemistry 220 (2021) 113436
Table 1
MIC50 and MIC90
Bocillin™,
a
fluorescent penicillin analog, using purified
(mg/mL) of 2 and comparator b-lactam antibiotics against a 198-
P. aeruginosa PBP3 according to an established protocol [37].
Increasing concentrations of 2 inhibited fluorescent labeling of the
protein by Bocillin™, with an IC50 of 5 ± 1
comparable to 1 (2.5 ± 0.5 M) and a previously published value for
doripenem (IC50 ¼ 2.3 ± 0.5 M, for Acinetobacter spp. PBP3) [37].
member panel of clinical isolates of Acinetobacter spp., as well as the 98-member
3
0
subset of carbapenem-resistant A. baumannii (CRAB). Chemical structures for all
agents are provided in Fig. 1.a See also Supplemental Table 2, page 19.
mM (Fig. 4). This value is
m
Compound
198 Acinetobacter
spp.
98 CRAB
m
MIC50
MIC90
MIC50
MIC90
2
.4. The compound 2 crystal structure complexed with
2
0.5
>32
64
8
16
8
>16
>32
>64
>64
>64
>64
1
>16
>32
>64
>64
>64
>64
P. aeruginosa PBP3a
Aztreonam (3)
Ceftazidime (4)
Meropenem (5)
Ceftazidime (4)/avibactam (8)
Ceftolozane (6)/tazobactam (7)
>32
>64
64
64
32
The crystal structure of P. aeruginosa PBP3 complexed to 2 was
determined to 2.0 Å resolution (Table 3). The difference electron
density in the active site shows that 2 has formed a covalent bond
with the catalytic S294 residue (Fig. 5). Density for most of the
compound 2 moieties is well resolved. This includes the chlorine
substituent on the aminothiazole ring, both amide groups, the
carboxyl group and the dihydropyrazole ring of the core. Note that
the occupancy for the chlorine atom refined to 0.58 whereas the
rest of the aminothiazole ring had an occupancy of 1.0. This
decreased occupancy for the chlorine atom is likely due to radiation
damage during the synchrotron radiation X-ray experiment.
Halogen-aromatic ring bonds are known to be sensitive to X-ray
radiation, and their breakage has been used to monitor radiation
damage in protein crystals [38]. Density for the 2-carboxypropan-
dimethyl moiety extending from the oxime and the 5,6-
a
MICs were determined using iron-depleted cation-adjusted Mueller-Hinton
broth that was supplemented with iron (as ferric chloride) as indicated. The initial
iron-depleted media was prepared by the standard treatment with cation-exchange
resin, which has been reported to reduce iron concentrations to 0.02
m
g/mL29
.
demonstrate that serum concentrations of sulbactam of 20
mg/mL
are readily attainable from intravenous dosing. Therefore, addi-
tional studies were undertaken to evaluate the in vitro effectiveness
of 2 under conditions of a constant level of sulbactam coadminis-
tration. The MIC values for 2 were determined using sulbactam set
at 20 mg/mL (Table 2). Under these conditions, the growth of only 3
of the isolates resistant to 2 were not significantly inhibited,
collectively implying that 98% of the full 198 member Acinetobacter
spp. panel could be susceptible to 2 when used in combination with
dihydroxyphthalimide siderophore group of
resolved in the electron density map indicating their inherent
flexibility (Fig. 5).
2 are not well
a 20 mg/mL coadministration of sulbactam.
The ligand 2 forms a number of hydrogen bonds in the active
site of P. aeruginosa PBP3 (Fig. 6). The chloroaminothiazole ring
hydrogen bonds with E291, and main chain oxygen and nitrogen
atoms of R489. The chloroaminothiazole also makes hydrophobic
interactions with G293, Y409, and A488. In addition, the chlorine
substituent makes a special close-to-linear “C-Cl / O interaction
with the carbonyl oxygen of Y407. Such favorable interactions with
a halogen atom have previously been observed in protein:ligand
interactions [39,40]. The amide side chain, which connects to the
aminothiazole ring, hydrogen bonds with N351 and the backbone
oxygen of T487. The carboxyl moiety of the core hydrogen bonds
with T487, S485, and conformation 2 of residue S349. The sec-
ondary nitrogen atom of the dihydropyrazole ring hydrogen bonds
with conformation 1 of S349 (Fig. 6). The amide moiety that serves
to attach the 5,6-dihydroxyphthalimide siderophore side chain of 2
2
.3. PBP inhibition
Based on the mechanism established previously for g-lactam 1,
was also suspected of inhibiting PBP3, an essential bacterial
transpeptidase. This was verified through labeling studies with
2
0
0
Table 2
MIC (
combined 1:1 (by mass), or when 2 was tested with a constant concentration of 10 at
g/mL. Individual MIC values for 2 and 10 are provided for comparison as well as
the identity of each Acinetobacter isolate. See text for details.
mg/mL) values in low iron media of 2 in combination with sulbactam, 10, when
20 m
Isolate No Strain Identitya
10
2 þ 10 (1:1) 2 þ 10 (20
2
mg/mL)
PR-314
PR-319
PR-325
PR-326
PR-340
PR-342
PR-345
PR-351
PR-362
PR-365
PR-380
PR-384
PR-387
PR-399
PR-401
PR-412
PR-423
PR-434
PR-452
PR-459
PR-464
PR-478
PR-482
PR-491
CRAB
CRAB
CRAB
CRAB
CSAB
CRAB
CRAB
CRAB
AN
>16 16
>16 >32
>16 32
4
4
ꢀ0.25
0.5
interacts with
a nearby water molecule (W#2). The 5,6-
>16
2
>16
dihydroxyphthalimide does not form many interactions in the
active site.
The compound 2 is chemically identical to the previously re-
ported 1, except the 5-position of the aminothiazole in 2 has a
chloro-substituent. The binding mode of 1 is very similar to that of
>16
>16
16
2
4
ꢀ0.25
ꢀ0.25
>16
ꢀ0.25
ꢀ0.25
ꢀ0.25
ꢀ0.25
ꢀ0.25
>16
ꢀ0.25
ꢀ0.25
ꢀ0.25
ꢀ0.25
ꢀ0.25
ꢀ0.25
ꢀ0.25
4
ꢀ0.25
ꢀ0.25
ꢀ0.25
ꢀ0.25
1
>32 >16
>16 16
>16 16
16
16
>16
>16
1
1
ꢀ0.25
ꢀ0.25
16
2
when bound to P. aeruginosa PBP3 (Fig. 7; PDBid 6VOT) [22]. A
AN
CRAB
CRAB
CRAB
CRAB
CRAB
AN
CRAB
CSAB
CSAB
CRAB
CSAB
CSAB
CRAB
AN
>16 16
>16 16
>16 16
>16 16
>16 16
difference is an inversion of the tertiary nitrogen in the dihy-
dropyrazole ring of 2 compared to 1. This is likely a consequence of
that the 2 PBP3 complex is determined to a higher resolution (2.0
vs. 2.5 Å resolution) allowing for improved refinement of this ring
region. A second difference is that residue E291 in the 2 PBP3
complex has moved closer to the aminothiazole ring compared to
the 1 PBP3 complex (Fig. 7).
16
16
16
8
1
8
2
>16
2
>16 16
>16
>16
16
>16
16
8
1
32
1
4
ꢀ0.25
8
2
.5. Pharmacokinetics and in vivo efficacy; drug-like attributes
ꢀ0.25
1
>16
0.5
>16 32
Pyrazolidinone 2 has many characteristics that are favorable
>16
2
drug-like attributes (details of the data are found in the Supporting
Information, pages 33e38) for potential administration either by IV
or inhaled routes as 2 has high solubility >100
phosphate buffer as measured by nephelometry. Though highly
a
Strain
identity:
CRAB
¼
carbapenem-resistant
CSAB ¼ carbapenem-susceptible A. baumannii; AN ¼ A. nosocomialis (carbapenem-
A.
baumannii;
mM in pH 7.5
susceptible).
5

J.A. Goldberg, V. Kumar, E.J. Spencer et al.
European Journal of Medicinal Chemistry 220 (2021) 113436
Fig. 4. Determination of the IC50 of 2 for P. aeruginosa PBP3 using a competitive assay. Bocillin™, a fluorescent substrate of PBP3 was reacted with enzyme that had been pre-
incubated with increasing concentrations of 2. An IC50 was calculated as the concentration of 2 required to reduce the fluorescence intensity of the Bocillin™-labeled protein
by 50%.
Table 3
microsomes is notable with a half-life >60 min in both prepara-
tions. Furthermore, 2 does not inhibit 7 of the 8 CYP enzymes it was
X-ray diffraction data collection and crystallographic refinement statistics for the
P. aeruginosa PBP3 complex with 2.
tested against at a 30
50% inhibition of CYP2C8 at 100
ated for cytotoxicity was not found when tested against human
primary hepatocytes at a maximal dose of 100 M.
In a general safety screen, testing 2 against 44 enzymes and
receptors the only activity of note was >50% inhibition at 30
against cyclooxygenase (COX-1) and phosphodiesterase PDE3A
Supporting Information, page 37). Testing against 6 ion channels,
including hERG at a 30 M maximal dose revealed 2 did not inhibit
mM concentration and showed approximately
Wavelength (Å)
0.97946
mM. Compound 2 was also evalu-
Resolution range (Å)
Space group
Unit cell (Å,
Total reflections
Unique reflections
Multiplicity
Completeness (%)
Mean I/sigma (I)
CC1/2
50.00e2.00 (2.03e2.00)
P2 2 2
1 1 1
67.77 82.66 88.80 90 90 90
427,156
m
ꢂ
)
mM
34,157 (1,673)
12.5 (12.6)
99.8 (99.8)
30.5 (4.4)
0.995 (0.934)
15.8 (115.9)
33.91e2.00 (2.05e2.00)
32,335 (2,169)
1,770 (141)
0.174 (0.202)
0.227 (0.237)
3,863
(
m
these channels (Supporting Information, page 38). The pharmaco-
kinetics (PK) of 2 was evaluated both subcutaneously and by
intravenous administration at a dose of 50 mg/kg in CD-1 mice
R-merge (%)
Resolution refinement (Å)
Reflections used in refinement
Reflections used for R-free
R-work
(Table 4). Both routes of dosing result in similar PK data. Notably,
the plasma levels of 2, at the 6h time point were >1.0 g/mL by both
m
R-free
dosing routes (Fig. 8 and Table 4). This value is clearly superior to 1
where at the 6-h timepoint its plasma concentration was about
Number of non-hydrogen atoms
Macromolecules
Ligand
3,621
49
0.35 mg/mL [22]. The cause for improved plasma level with 2 is not
Solvent
Protein residues
RMS(bonds, Å)
RMS(angles,
Ramachandran favored (%)
Ramachandran allowed (%)
Ramachandran outliers (%)
193
473
0.010
1.65
97.64
2.15
0.22
known, though it might be due to clearance differences caused by a
slight increase in protein binding or a small increase in LogP due to
the additional chlorine atom. Nevertheless, the >1.0 mg/mL plasma
level encouraged us to comparatively test both 2 and 1 in a mouse
efficacy model as this plasma level is above the MIC50 values of 2 for
both P. aeruginosa and A. baumannii.
ꢂ
)
We chose AR-BANK#0229 strain of carbapenem-resistant
P. aeruginosa as the infectious agent for the neutropenic thigh
infection model. This decision was made for several reasons: a) this
strain establishes infection in mice; b) both compound 2, and 1
Statistics for the highest-resolution shell are shown in parentheses.
have potent activity against this strain (MIC ¼ 0.5
mg/ml and
0
.25 g/mL, respectively); and c) the strain is highly resistant to
m
currently clinically used antibiotics as it is susceptible to only
colistin, amikacin, and gentamicin (Supplemental Table 4, page 42).
Neutropenia was induced with cyclophosphamide. Animals
6
were then intramuscularly (IM) inoculated with 1.02 ꢃ 10 CFU/
mouse (0.1 mL/animal) of the P. aeruginosa AR-BANK#0229 strain.
Test antibiotics, 1 and 2, both at 50 and 100 mg/kg, were admin-
istered subcutaneously (SC) four times per day (QID) with 6 h in-
tervals (q6h) at 2, 8, 14 and 20 h after infection. The reference
control was colistin, which was dosed at 30 mg/kg and adminis-
tered subcutaneously (SC) twice (BID) with 12 h intervals (q12h) at
2
and 14 h after infection. Animals were sacrificed at 2 or 26 h post-
infection, and the thigh tissues were harvested and weighed from
each of the test animals. The bacterial counts (CFU/g) of thigh tissue
homogenates were compared. Full details of the method and pro-
tocol are in the Supplemental Information as well as the individual
animal data (Supplemental Information, pages 45e57). Bacterial
burden (CFU/g tissue) of test article treated animal groups was
compared to the baseline bacterial count at 2 h after infection and
the difference in counts (D) was reported. The significance of effects
was assessed with ANOVA. The burden was also compared to the
vehicle control.
Fig. 5. Omit Fo-Fc electron density showing the presence of a covalently bound 2. The
compound 2 is shown with cyan-colored carbon atoms. The density is contoured at the
3
s level.
soluble, 2 has poor Caco-2 permeability and would not be expected
to have oral bioavailability thus IV and sub cutaneous PK was ob-
tained (vide infra). Compound 2 is highly protein-bound as shown
by measurements in both mouse and human plasma, 94% and 88%,
respectively. Stability of 2 to both human and CD-1 mouse
6

J.A. Goldberg, V. Kumar, E.J. Spencer et al.
European Journal of Medicinal Chemistry 220 (2021) 113436
Fig. 6. Stereo diagram depicting the interactions of 2 in the active site of P. aeruginosa PBP3. Hydrogen bonds are depicted as dashed lines.
2.6. Resistance
Although 2 showed broad coverage against a large panel of
resistant clinical isolates of Acinetobacter spp. including
carbapenem-resistant A. baumannii, a fraction of all tested strains
(24 of 198, 12%) and the carbapenem-resistant subgroup (15 of 98,
1
5%) demonstrated MICs ꢁ16
m
g/mL.
Understanding the mechanism or mechanisms of resistance for
is important as it allows the optimization of partner agents or
2
alternative dosing schedules to ameliorate potential issues. This
knowledge will help predict whether further strains will be sus-
ceptible and influence future clinical use, as well as design and
synthesis of subsequent analogs.
There are a number of mechanisms that have been identified for
resistance to antibiotics for Acinetobacter spp. as well as other
Gram-negative pathogens. They fall into 4 general categories: 1)
degradation of the antibiotic (e.g.,
b-lactamase hydrolysis); 2)
target modification (e.g., PBP mutations or changes in the expres-
sion of PBP genes); 3) efflux of the antibiotic from the periplasm or
cytoplasm (e.g., mutations or decreased changes in expression of
pump genes such as ErmAB-TolC, macA, UadeA-H, mdtABC); or 4)
decreased permeability of the bacterial outer membrane (e.g., TolC,
OmpH, OPRD genes). All of these represent potential mechanisms
for resistance to 2. Additionally, due to the incorporation of the
siderophore moiety as a key component of the antibiotic molecule,
changes in the expression of genes related to the bacterial iron
transport system could also have an effect on antibiotic suscepti-
bility as was identified as a potential resistance mechanism for
cefiderocol, 9 [Ito, A. et al. IDWeek 2018 poster 696. https://idsa.
confex.com/idsa/2018/webprogram/Paper69661.html].
Fig. 7. Superpositioning of the 1 and 2 bound structures of P. aeruginosa PBP3. The
carbon atoms of the 1:PBP3 complex is shown in salmon color; while 2 is colored cyan
with its protein shown in white.
Table 4
Individual plasma concentrations of 2 after sc (50 mg/kg) dosing in mice, each value
represents an individual mouse, showing mean blood levels >1
m
g/mL at the 8-h
time point.
Time (h)
Sample Conc (ng/ml)
Mean (ng/ml)
SD (ng/ml)
0
0
1
2
4
6
8
2
.167
.5
48690
20245
3138
3273
1534
1426
1001
678
48689
14355
3515
2876
1732
1102
1473
545
44921
14094
3186
3072
739
1425
1126
579
47433
16231
3280
3074
1751
1318
1200
601
2176
3478
205
199
526
187
245
69
2.7. Genomic analysis for resistance genes
4
Our initial investigation to determine the mechanism of resis-
tance toward 2 seen in Acinetobacter spp., as well as P. aeruginosa
and K. pneumoniae, was undertaken by comparing the genomes of
susceptible and resistant organisms. Using whole-genome
As might have been predicted by the pharmacokinetic results, 1
was not found to have efficacy in the animal model against this
carbapenem-resistant strain even though the MIC values indicated
the strain had good susceptibility of 0.25 mg/mL (Fig. 9). Impor-
tantly, 2, with superior pharmacokinetics does show a dose-
dependent reduction in CFU values against this difficult to treat
P. aeruginosa strain. It is notable that the MIC value for 2 of 0.5 mg/
mL is below the expected blood levels of 2 at the 6 h dosing interval
and, in spite of the high protein binding of 94%, encouraging ac-
tivity was seen (Fig. 9).
sequencing (WGS), potential resistance genes including
b-lacta-
mases, efflux pumps, porin proteins, PBPs, and iron transport were
analyzed for potential gene mutations [41] (see supplemental
genomic analysis data for details). There were no identified trends
in the presence of resistance genes related to b-lactamases, efflux
pumps, outer membrane porins, or iron transport when comparing
isolates of all analyzed species. When analyzing the PBP genes of
P. aeruginosa, a mutation in the PBP3, F533L, previously identified
as a gain of function mutation for
b-lactam antibiotics [42,43] may
be partially responsible for resistance to 1 and 2 (Table 5). The side
7

J.A. Goldberg, V. Kumar, E.J. Spencer et al.
European Journal of Medicinal Chemistry 220 (2021) 113436
Fig. 8. The mean plasma concentration-time profile for YU253911, 2, after sc dosing (50 mg/kg) in mice.
b
-lactamases NDM-1, VIM-2, and IMP-1, however, did demonstrate
measurable activity (Table 6). Remarkably, the calculated catalytic
ꢄ1 ꢄ1
were orders of magni-
efficiencies, kcat/K
m
of 0.003e0.02
mM
s
tude lower than those previously determined for a comparator
ꢄ1 ꢄ1
antibiotic (imipenem 2.7e6.7
was previously reported for
Furthermore, the observed stability to purified
m
M
s
) [22] and similar to what
ꢄ1 ꢄ1
) [22].
g
-lactam 1 (0.02e0.07
mM
s
b
-lactamase en-
zymes correlates with the MIC data, where a relationship was not
noted between microbiologic activity and the presence or absence
of any b-lactamase gene. See Supplemental Table 3, page 27, for the
full listing of the Acinetobacter spp.
b-lactamase genes in this
experiment.
2.9. Efflux inhibitor experiments
To further investigate potential resistance mechanisms, the
growth inhibition of 2 in combination with efflux pump inhibitors
EPIs) was examined. The MIC ( g/mL) values of 2 in combination
(
m
Table 5
MICs (
m
g/mL) of
g
-lactams 1 and 2 against representative susceptible (MIC ꢀ4
mg/
mL) and resistant (MIC ꢁ16
m
g/mL) clinical isolates of P. aeruginosa that have been
Fig. 9. The test compounds YU253434 1, YU253911 2, and colistin efficacy in the
P. aeruginosa AR-BANK#0229 thigh infection model.
whole genome sequenced and the identity of the amino acid at position 533:
phenylalanine (wild-type) or leucine (mutation shown previously to provide gain of
(
*) A significant difference (p < 0.05) between the vehicle control and treatment group
function for b-lactam antibiotics in some resistant strains of P. aeruginosa).
was determined by one-way ANOVA followed by Dunnett’s test.
Error bars are SEM values.
P. aeruginosa ID no.
1
2
Position 533 residue
PR-545
PR-548
PR-567
PR-672
PR-604
PR-638
PR-676
PR-503
PR-564
PR-606
PR-627
PR-635
PR-636
PR-651
PR-668
PR-673
PR-677
PR-680
PR-688
PR-699
ꢀ 0.25
ꢀ 0.25
ꢀ 0.25
ꢀ 0.25
ꢀ 0.25
>16
16
16
>16
16
0.5
1
ꢀ 0.25
ꢀ 0.25
ꢀ 0.25
0.5
Phenylalanine
Phenylalanine
Phenylalanine
Phenylalanine
Phenylalanine
Phenylalanine
Phenylalanine
Phenylalanine
Leucine
Phenylalanine
Leucine
Phenylalanine
Leucine
chain of F533 in the active site of P. aeruginosa PBP3 makes contacts
with the
Fig. 6).
g-lactam acylation product as seen in the X-ray structure
(
2
16
2.8. b-Lactamase stability
>16
>16
>16
16
>16
>16
>16
>16
16
As was previously reported, the dihydroxyphthalimide side-
rophore mimetic side chain appended to the -lactam core was: a)
intended to impart stability against -lactamase hydrolysis; and b)
promote periplasm uptake. Thus, -Lactam 2 was evaluated as a
potential substrate against representative members of all 4 Ambler
classes of -lactamases. We did not observe measurable reactions
>16
16
g
>16
>16
>16
>16
16
>16
>16
>16
b
g
Leucine
Leucine
Leucine
Phenylalanine
Leucine
b
with KPC-2 (class A K. pneumoniae carbapenemase), ADC-7 (class C
Acinetobacter-derived cephalosporinase) or OXA-23 (class D oxa-
cillinase) when tested as purified isolated enzymes. Class B metallo
>16
>16
>16
Leucine
Phenylalanine
8

J.A. Goldberg, V. Kumar, E.J. Spencer et al.
European Journal of Medicinal Chemistry 220 (2021) 113436
Table 6
Susceptibility of 2 to
4.1.1. Synthesis of (S,Z)-6-(2-(((1-(tert-butoxy)-2-methyl-1-
oxopropan-2-yl)oxy)imino)-2-(5-chloro-2-(tritylamino)thiazol-4-
yl)acetamido)-2-((2-(5,6-dihydroxy-1,3-dioxoisoindolin-2-yl)ethyl)
carbamoyl)-5-oxo-6,7-dihydro-1H,5H-pyrazolo[1,2-a]pyrazole-3-
carboxylic acid
b
-Lactamase hydrolysisa.
ꢄ1
ꢄ1 ꢄ1
s )
Metallo-
b
-lactamase
kcat (s
)
K
m
(
mM)
k
cat/K
m
(
mM
NDM-1
VIM-2
IMP-1
KPC-2
ADC-7
OXA-23
35 ± 4
54 ± 6
5 ± 1
NM
1808 ± 200
4400 ± 450
1490 ± 300
NM
0.020 ± 0.002
0.012 ± 0.001
0.003 ± 0.001
NM
(Z)-2-(((1-(tert-butoxy)-2-methyl-1-oxopropan-2-yl)oxy)
imino)-2-(5-chloro-2-(tritylamino)thiazol-4-yl)acetic acid [44] 15
NM
NM
NM
NM
(
537 mg, 0.89 mmol) in dry dichloromethane (7 mL) and catalytic
DMF was cooled in an ice bath and treated with oxalyl chloride
484 L of 2.0 M solution, 0.97 mmol) and stirred for 1 h. In a second
NM
NM
a
Steady-state reactions of 2 were monitored using purified enzymes: KPC-2,
ADC-7, OXA-23, NDM-1, VIM-2, and IMP-1. Kinetic parameters provided were
determined from double reciprocal plots (Supplemental Figure 2, page 44).
Measurable reaction was not detected for KPC-2, ADC-7, or OXA-23 using 200 nM
(
m
flask starting amine 14 as the TFA salt (348 mg, 0.81 mmol) in
dichloromethane (7 mL) was cooled in an ice bath and MSTFA
enzyme concentration and 100
m
M 2. NM ¼ not measureable.
(600 mL, 3.2 mmol) and Hunig’s base (773 mL, 4.4 mmol) was added.
After stirring for 20 min all of the starting amine was in solution.
During this time the dichloromethane from the acid chloride
forming reaction was evaporated and placed on a high vacuum to
give a colorless foam. This foam was dissolved in dry DCM (7 mL)
and added to the MSTFA treated amine followed by the addition of
with the EPIs carbonyl cyanide 3-chlorophenylhydrazone (CCCP) or
phenylalanine-arginine -naphthylamide (Pa N) were tested
against a subgroup of Acinetobacter spp. isolates resistant to 2
Supplemental Table 4, page 32). The EPIs were kept at a constant
concentration at levels commonly used in published reports against
Gram-negative bacteria (2 g/mL for CCCP and 32 g/mL for PA N).
b
b
(
Hunig’s base (250
to room temperature over 1 h and stirred overnight to give the
crude product (S,Z)-6-(2-(((1-(tert-butoxy)-2-methyl-1-
mL, 1.4 mmol). The reaction was allowed to warm
m
m
b
No significant inhibition effect on growth was observed directly
from the EPIs, and no consistent effect on MIC values were observed
when combined with 2. Potential synergistic effects were observed
for only one isolate, PR-452, when combined with CCCP and since
this was not a general effect, a detailed investigation was not
pursued.
oxopropan-2-yl)oxy)imino)-2-(2-(tritylamino)thiazol-4-yl)acet-
amido)-2-((2-(5,6-dihydroxy-1,3-dioxoisoindolin-2-yl)ethyl)car-
bamoyl)-5-oxo-6,7-dihydro-1H,5H-pyrazolo
[1,2-a]pyrazole-3-
carboxylic acid that was not isolated but directly deprotected in
situ.
4.1.2. Synthesis of (S,Z)-6-(2-(2-amino-5-chlorothiazol-4-yl)-2-
(((2-carboxypropan-2-yl)oxy)imino)acetamido)-2-((2-(5,6-
dihydroxy-1,3-dioxoisoindolin-2-yl)ethyl)carbamoyl)-5-oxo-6,7-
dihydro-1H,5H-pyrazolo[1,2-a]pyrazole-3-carboxylic acid (2)
To the crude material from the overnight reaction above (0.82 g,
3
. Conclusions
In conclusion, we demonstrate the potency and effectiveness of
0
.81 mmol) was added triethyl silane (0.64 mL, 4.0 mmol). The
solution was cooled in and ice bath and trifluoroacetic acid (6.2 mL,
1 mmol) was added. The ice bath was removed after 30 min and
the reaction was stirred at room temperature for 1 h. Toluene
25 mL) was added and the reaction was evaporated to dryness. The
a novel
g-lactam siderophore antibiotic. Our intent was to build
upon previous investigations performed with compound 1. We
show potency against MDR strains of K. pneumoniae, Acinetobacter
spp., and P. aeruginosa (MIC50 ꢀ 0.5
Consistent with these microbiological findings, molecular analyses
reveal stability against problematic -lactamases. The atomic
structure of P. aeruginosa PBP-3 at 2.0 Å resolution revealed an
important “C-Cl / O interaction with the carbonyl oxygen of Y407.
PK/PD and animal testing established that reductions in CFU were
significant when compared to colistin. Importantly, toxicity was not
evident in a series of assays. Against CRAB, we lowered MICs
8
mg/ml vs. > 8 for meropenem).
(
crude reaction mixture was dissolved in dimethyl sulfoxide, diluted
with water and chromatographed using reverse phase C18 MPLC
eluting with 0e40% acetonitrile with 0.1% formic acid in water with
b
0
0
0.1% formic acid to give the product (S,Z)-6-(2-(2-amino-5-
chlorothiazol-4-yl)-2-(((2-carboxypropan-2-yl)oxy)imino)acet-
amido)-2-((2-(5,6-dihydroxy-1,3-dioxoisoindolin-2-yl)ethyl)car-
bamoyl)-5-oxo-6,7-dihydro-1H,5H-pyrazolo
carboxylic acid, 2, as yellow powder after lyophilization
139 mg, 23%). H NMR (400 MHz, DMSO‑d 12.61 (s,1H),10.34 (s,
[1,2-a]pyrazole-3-
significantly by combining 2 with sulbactam, designing a novel g-
lactam BLI combination. Studies are planned to further increase our
understanding regarding optimal structure activity relationships
a
1
(
6
) d
4
4
H), 8.78 (d, J ¼ 8.34 Hz, 1H), 8.56e7.91 (m, 1H), 7.38 (s, 2H), 7.12 (s,
(
SARs) and dosing to overcome resistant infection.
H), 5.03 (dt, J ¼ 8.05, 10.89 Hz, 1H), 4.11 (d, J ¼ 12.50 Hz, 1H), 3.81
(
1
dd, J ¼ 6.81, 10.26 Hz, 2H), 3.67e3.48 (m, 2H), 2.96 (dd, J ¼ 8.71,
þ
0.98 Hz, 1H), 1.66e1.36 (m, 6H). Mass spectrum MþH ¼ 721.0;
HRMS (ESI/QTOF) Calcd for C27
H25Cl
1
N
8
O
12
S
1
720.1001, found
4
. Experimental
þ
MþH 721.1077.
4.1. Syntheses
4.2. Minimum inhibitory concentrations, MICs
The reagents and solvents used for synthesis were of reagent
grade quality. Dry solvents were purchased and used as such. All
compounds were individually purified by chromatography on silica
gel or by recrystallization and were of >95% purity for character-
ization purposes as determined by LCMS using UV absorption at
Bacterial strains used were from previously described collec-
tions [22,45]. MICs were determined using the general recom-
mendations of the Clinical and Laboratory Standards Institute
(CLSI). Standard broth microdilution methods were followed but
4
2
20 or 280 nM and/or NMR integration. In practice, compounds
with a slightly lower inoculum (6 ꢃ 10 cfu/mL) which afforded no
were not always purified to >95% purity prior to using in the next
synthetic step, and often crude material was of sufficient purity and
was carried forward. Copies of the spectra of 2 are included in the
Supplemental Information, page 8.
difference in MICs in our testing. MICs were performed using iron-
depleted cation-adjusted Mueller-Hinton broth, except as
mentioned elsewhere, using a standard protocol used with other
siderophore-containing antibiotics.
9

J.A. Goldberg, V. Kumar, E.J. Spencer et al.
European Journal of Medicinal Chemistry 220 (2021) 113436
4
.3. Protein expression, purification, crystallization, and structure
using this same method, resulting in similar values to those pre-
viously reported [22] (Supplemental Figure 1, page 43 and
Supplemental Table 5, page 44).
determination of the compound 2 P. aeruginosa PBP3 complex
The P. aeruginosa PBP3 protein was expressed, purified, and
crystallized as previously described [22]. A crystal of P. aeruginosa
PBP3 was soaked with 2 mM compound 2 for 43 h in mother liquor
before freezing the crystal in liquid nitrogen prior to data collection.
Data were collected at the SSRL synchrotron beamline 12-2 and
processed to 2.0 Å resolution using HKL3000 [46]. The structure
was solved using molecular replacement using PHASER [47] with
the PBP3 ceftazidime complex protein coordinates as the search
model (PBD ID 3PBO). The structure was refined using REFMAC 5
4.6. Genomic analysis
Acinetobacter spp., and K. pneumoniae sequences were down-
loaded from the sequencing read archive project PRJNA384060,
PRJNA384065, and PRJNA339843, respectively (National Center for
Biotechnology Information, U.S. National Library of Medicine, 8600
Rockville Pike, Bethesda MD, 20894 USA). Pseudomonas aeruginosa
were sequenced by the Illumina Hiseq platform with 2x150bp
paired-end sequencing. Reads were assembled and annotated us-
ing PATRIC, the Pathosystems Resource Integration Center [41].
Antibiotic resistant genes were identified using RES Finder (Center
for Genomic Epidemiology; http://www.genomicepidemiology.
org/). Amino acid variations were identified by comparison of
strains to the Acinetobacter ATCC 17978 and ATCC 19606, Pseudo-
monas aeruginosa PA01, or Klebsiella pneumoniae MGH 78578
(GenBank Accessions CP000523, ACQB00000000, CP001183,
CP000647).
[
48], and the program Coot [49] was used for model building.
Refinement parameter files for compound 2 were generated using
ACEDRG [50]. Molecular figures were generated using Pymol
(
www.pymol.org). The coordinates and structure factors of the
P. aeruginosa PBP3 YU253911 complex have been deposited with
the Protein Data Bank (PDB ID 7LC4).
4.4. PBP binding kinetics
A method was adapted from the work using purified, soluble
P. aeruginosa PBP3 and Bocillin™, a fluorescent
strate [37]. Reactions were conducted in 10 mM phosphate-
buffered saline at pH 7.4 using 1.6 M P. aeruginosa PBP3 incu-
b
-lactam and sub-
Accession codes
m
PDB code for P. aeruginosa PBP3 with bound YU253911, 2, is PDB
ID 7LC4. Authors will release the atomic coordinates upon article
publication.
bated with increasing concentrations of 2. To ensure that equilib-
rium between 2 and PBP3 occurred, the enzyme was preincubated
ꢂ
with the compound for 20 min at 37 C before the addition of 50
m
M
Bocillin™ and then incubated for an additional 20 min. The re-
actions were stopped by adding SDS-PAGE loading dye and boiling
for 2 min. Samples were analyzed by SDS-PAGE and the gel illu-
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
minated at
l
¼ 365 nm and imaged with a Fotodyne gel imaging
system. ImageJ analysis software was used to assign fluorescence
intensity (FI). The IC50 was calculated as the concentration of 2
required to reduce the FI of the Bocillin™-labeled protein by 50%.
Acknowledgements
4
.5.
b
-lactamase stability testing
MSP: Financial support was provided by the Program in Inno-
vative Therapeutics for Connecticut’s Health grant by the Con-
necticut Bioscience Innovation Fund, administered by the
Connecticut Innovations. RAB: Research reported in this publica-
tion was supported by the National Institute of Allergy and Infec-
tious Diseases of the National Institutes of Health (NIH) under
Award Numbers R01AI100560, R01AI063517, and R01AI072219.
This study was also supported in part by funds and/or facilities
provided by the Cleveland Department of Veterans Affairs, Award
Numbers 1I01BX002872 to KMP-W and 1I01BX001974 to RAB, from
the Biomedical Laboratory Research & Development Service of the
VA Office of Research and Development, and the Geriatric Research
Education and Clinical Center VISN 10 (RAB). The content is solely
the responsibility of the authors and does not necessarily represent
the official views of the NIH or the Department of Veterans Affairs.
BNK: Research reported in this publication was supported by a
grant from the National Institutes of Health, R01AI090155.
RAB wishes to gratefully thank the Antimicrobial Resistance
Leadership Group (ARLG) for the use of the isolates. The research
described in this publication is supported, at least in part, by a grant
from the National Institutes of Health through Duke University and
the findings, opinions, and recommendations expressed herein are
those of the authors and not necessarily those of Duke University or
the National Institutes of Health. This study is also supported by the
National Institute of Allergy and Infectious Diseases of the National
Institutes of Health (NIH) under Award Number UM1AI104681. This
work utilized NIAID’s suite of preclinical services for in vitro and
in vivo assessments (Contract Nos. 75N93019D00022/
75N93020F00001 and HHSN272201700020I/75N93020F00159).
Steady-state kinetics were determined with purified enzymes
KPC-2, ADC-7, OXA-23, NDM-1, VIM-2, and IMP-1) using an Agilent
(
model 8453 diode array spectrophotometer as previously described
ꢂ
[
22]. Assays were performed at 25 C (room temperature) using
1
0 mM PBS, pH 7.4 (KPC-2, ADC-7), 50 mM sodium phosphate
buffer supplemented with 20 mM sodium bicarbonate (OXA-23) or
0 mM HEPES, pH 7.5, 0.2 M NaCl, 50 g/ml bovine serum albumin,
and 50 M Zn (NDM-1, VIM-2, and IMP-1).
1
m
m
Compound 2 was used as a substrate at excess molar concen-
trations to establish pseudo-first-order kinetics. The following
ꢄ1
ꢄ1
extinction coefficient was used: 2,
velocity determinations, a 0.2 cm path length quartz cuvette was
employed. Hydrolysis of 600 M 2 was monitored over time until
completion. Based on the starting absorbance (
¼ 336 nm, 600
) and final absorbance ( M 2), concentrations of 2
¼ 336 nm, 0
were determined along the progress curve and velocities during a
0 s period for each concentration calculated. A double reciprocal
plot (1/[S] vs. 1/V) was employed to obtain the steady-state kinetic
parameters Vmax, kcat, and K according to Equations (1) and (2):
De336 ¼ ꢄ4362 M cm . For
m
l
mM
2
l
m
1
m
1
/V ¼ K
m
/Vmax(1/[S]) þ 1/Vmax
Eq. 1
k
cat ¼ Vmax/[E]
Eq. 2
where V ¼ reaction velocity, K
m
¼ Michaelis-Menten constant,
V
max ¼ maximum reaction velocity, and [S] ¼ substrate concen-
tration. Compound 1 was retested with NDM-1, VIM-2, and IMP-1
10

J.A. Goldberg, V. Kumar, E.J. Spencer et al.
European Journal of Medicinal Chemistry 220 (2021) 113436
FVDA: We also thank Stanford Synchrotron Radiation Light-
source (SSRL) for help with data collection. Use of the SSRL, SLAC
National Accelerator Laboratory, is supported by the U.S. Depart-
ment of Energy, Office of Science, Office of Basic Energy Sciences
under Contract No. DE-AC02-76SF00515. The SSRL Structural Mo-
lecular Biology Program is supported by the DOE Office of Biological
and Environmental Research, and by the National Institutes of
Health, National Institute of General Medical Sciences (including
P41GM103393). The contents of this publication are solely the re-
sponsibility of the authors and do not necessarily represent the
official views of NIGMS or NIH.
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Appendix A. Supplementary data
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