Identification of a bacteria-produced benzisoxazole with antibiotic activity against multi-drug resistant Acinetobacter baumannii

Article abstract of DOI:10.1038/s41429-021-00412-7

The emergence of multi-drug resistant pathogenic bacteria represents a serious and growing threat to national healthcare systems. Most pressing is an immediate need for the development of novel antibacterial agents to treat Gram-negative multi-drug resistant infections, including the opportunistic, hospital-derived pathogen, Acinetobacter baumannii. Herein we report a naturally occurring 1,2-benzisoxazole with minimum inhibitory concentrations as low as 6.25 μg ml^?1 against clinical strains of multi-drug resistant A. baumannii and investigate its possible mechanisms of action. This molecule represents a new chemotype for antibacterial agents against A. baumannii and is easily accessed in two steps via de novo synthesis. In vitro testing of structural analogs suggest that the natural compound may already be optimized for activity against this pathogen. Our results demonstrate that supplementation of 4-hydroxybenzoate in minimal media was able to reverse 1,2-benzisoxazole’s antibacterial effects in A. baumannii. A search of metabolic pathways involving 4-hydroxybenzoate coupled with molecular modeling studies implicates two enzymes, chorismate pyruvate-lyase and 4-hydroxybenzoate octaprenyltransferase, as promising leads for the target of 3,6-dihydroxy-1,2-benzisoxazole.

Full text of DOI:10.1038/s41429-021-00412-7

The Journal of Antibiotics (2021) 74:370–380
https://doi.org/10.1038/s41429-021-00412-7
ARTICLE
Identification of a bacteria-produced benzisoxazole with antibiotic
activity against multi-drug resistant Acinetobacter baumannii
2
Robert W. Deering1 Kristen E. Whalen
Ivan Alvarez1 Kathryn Daffinee3,4 Maya Beganovic3,4
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Kerry L. LaPlante3,4 Shreya Kishore2 Sijing Zhao2 Brent Cezairliyan5 Shen Yu5 Margaret Rosario1
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Tracy J. Mincer6 David C. Rowley
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Received: 29 October 2020 / Revised: 21 December 2020 / Accepted: 4 January 2021 / Published online: 12 February 2021
© The Author(s) 2021. This article is published with open access
Abstract
The emergence of multi-drug resistant pathogenic bacteria represents a serious and growing threat to national healthcare
systems. Most pressing is an immediate need for the development of novel antibacterial agents to treat Gram-negative multi-
drug resistant infections, including the opportunistic, hospital-derived pathogen, Acinetobacter baumannii. Herein we report
a naturally occurring 1,2-benzisoxazole with minimum inhibitory concentrations as low as 6.25 μg ml−1 against clinical
strains of multi-drug resistant A. baumannii and investigate its possible mechanisms of action. This molecule represents a
new chemotype for antibacterial agents against A. baumannii and is easily accessed in two steps via de novo synthesis. In
vitro testing of structural analogs suggest that the natural compound may already be optimized for activity against this
pathogen. Our results demonstrate that supplementation of 4-hydroxybenzoate in minimal media was able to reverse 1,2-
benzisoxazole’s antibacterial effects in A. baumannii. A search of metabolic pathways involving 4-hydroxybenzoate coupled
with molecular modeling studies implicates two enzymes, chorismate pyruvate-lyase and 4-hydroxybenzoate octaprenyl-
transferase, as promising leads for the target of 3,6-dihydroxy-1,2-benzisoxazole.
Introduction
These authors contributed equally: Robert W. Deering, Kristen
E. Whalen
Acinetobacter baumannii is a non-fermenting, Gram-
negative member of the Gammaproteobacteria commonly
implicated in nosocomial sepsis, wound infections, and
ventilator-associated pneumonia [1]. The World Health
Organization recognizes antibiotic resistance as one of the
three greatest threats to human health, and recently classi-
fied carbapenem-resistant A. baumannii as one of the most
urgent threats, calling for a renewed investment in antibiotic
development [2]. A. baumannii tenacity in hospitals is
enhanced both by its ability to develop antibiotic resistance
and capacity to survive on surfaces, including skin, for
several days [3]. A. baumannii can also spread via aero-
solization, promoting this pathogen’s ability to easily
colonize new environments [4–6]. A. baumannii is a
member of the so-called ‘ESKAPE’ pathogens [7] and has
garnered significant attention due to its propensity to cause
wound infections in U.S. service members who served in
Iraq and Afghanistan [8]. Alarmingly, intensive care unit
patients contracting A. baumannii infections have a high
mortality rate (>50%), complicated by the fact that new
pan-drug resistant (PR) strains of A. baumannii with
Supplementary information The online version contains
supplementary material available at https://doi.org/10.1038/s41429-
021-00412-7.
* Kristen E. Whalen
kwhalen1@haverford.edu
* Tracy J. Mincer
tmincer@fau.edu
* David C. Rowley
drowley@uri.edu
1
Department of Biomedical and Pharmaceutical Sciences, College
of Pharmacy, University of Rhode Island, Kingston, RI, USA
2
Department of Biology, Haverford College, Haverford, PA, USA
3
Department of Pharmacy Practice, College of Pharmacy,
University of Rhode Island, Kingston, RI, USA
4
Infectious Diseases Research Program, Providence Veterans
Affairs Medical Center, Providence, RI, USA
5
Octagon Therapeutics, Inc., Cambridge, MA, USA
6
Wilkes Honors College and Harbor Branch Oceanographic
Institute, Florida Atlantic University, Boca Raton, FL, USA

Identification of a bacteria-produced benzisoxazole with antibiotic activity against multi-drug. . .
371
resistance to all chemotherapeutic treatment options have
now emerged [1, 8–10]. Carbapenems, typically in combi-
nation with other antimicrobial agents (e.g., the polypeptide
colistin) are a commonly utilized treatment option for multi-
drug resistant (MDR) A. baumannii, but it is
estimated that more than half of MDR strains are now
carbapenem-resistant and colistin has significant tox-
icological and dosing concerns [1, 11, 12]. With the lack of
new antibiotics in the drug discovery pipeline to treat Gram-
negative infections, coupled with accelerated evolution
of antibiotic resistance, it is imperative that new anti-
bacterial drugs for treating A. baumannii infections be
developed [13].
Although uncommonly reported from natural sources,
benzisoxazole scaffolds are well represented in pharmacol-
ogy, having shown significant importance in anti-HIV [14],
antimicrobial [15–18], antipsychotic [19, 20], anti-
inflammatory [17, 21, 22], antioxidation [17, 23], anticancer
[23–26], anticonvulsant [27] and antidiabetic [28, 29]
research. Moreover, 1,2-benzisoxazole derivatives, which
include zonisamide, risperidone, paliperidone, and iloper-
idone, are all FDA approved and currently in use for epilepsy,
mood disorders, and schizophrenia, respectively [30–33].
Herein we report the identification of 3,6-dihydroxy-1,2-
benzisoxazole (1), a potent antibiotic against A. baumannii
produced by a marine bacterium identified as a Bradyrhizo-
bium denitrificans. A series of synthetically prepared analogs
define key structural features for the antibacterial effects. We
further provide evidence suggesting the mechanism of action
(MOA) of 3,6-dihydroxy-1,2-benzisoxazole. In discovering
4-hydroxybenzoate (4-HB)’s ability to reverse the anti-
bacterial property of 1, we investigate two possible 4-HB-
utilizing target enzymes, chorismate pyruvate-lyase (CPL)
and 4-HB octaprenyltransferase.
from 72 l of B158 microbial culture resulted in the isolation
of compound 1.
Compound 1 was isolated as an amorphous white pow-
der. An [M − H]− ion of 150.0204 using HRESIMS indi-
cated a molecular formula of C7H5NO3. 1H NMR
resonances at δH 7.49, 6.75, and 6.67 as well as the mole-
cular formula suggested 1 to be 3,6-dihydroxy-benzisox-
azole [35]. Because only 1 mg of compound 1 was isolated
from 14.1 g of crude extract, 3,6-dihydroxy-benzisoxazole
was synthesized (Fig. S1) and determined to be identical to
the natural product by NMR spectroscopy (Table S1) [35].
The synthetic derivative of compound 1 showed potent
antimicrobial activity against Escherichia coli MDR strains
AG102, MG1655 ΔBC/pABM, and MG1655 ΔBC/pXYM
with an MIC between 0.31 and 0.63 µg ml−1
.
3,6-Dihydroxy-1,2-benzisoxazole (1) inhibits the
growth of clinically relevant strains of A. baumanii
Compound 1 was previously reported to possess growth
inhibitory effects against Gram-negative pathogenic bac-
teria, but not Gram-positives [36]. Because this prior testing
was completed more than 30 years ago and did not include
A. baumannii, we tested 1 for antibacterial activity against a
panel of four clinically relevant A. baumannii strains
(Table S2). Two strains (L1051 and NR-13382) were
classified as carbapenem-resistant A. baumannii (CRAB)
and as multi-drug resistant (MDR) organisms by having
non-susceptibility to one or more agents in three or more
antimicrobial categories using previously published guide-
lines [37, 38]. Minimum inhibitory concentrations (MICs)
were determined to be in the range of 6.25 μg ml−1 to 50 μg
ml−1 with the most potent antibacterial effects against the
MDR strains NR-13382 and L1051 (6.25 and 12.5 μg ml−1
,
respectively). While the MICs for these clinical strains were
measured in Muller-Hinton broth (MHB), additional MIC
testing was pursued using both MHB and DM01, a defined
minimal medium supplemented with sodium pyruvate as a
carbon source. We hypothesized that nutrient replete media
could contain compounds that antagonized the antibiotic
activity of 1, which could provide insight into compound
1’s MOA. Interestingly, compound 1 was much more potent
against multiple strains of Pseudomonas aeruginosa,
E. coli, A. baumannii, and Klebsiella pneumoniae in assays
using minimal media, DM01 (Table 1).
Results
Bioassay-guided fractionation and dereplication of
3,6-dihydroxy-1,2-benzisoxazole
Crude organic extract from the exudate of B. denitrificans
(Isolate B158) was generated and initially screened in the
p-iodonitrotetrazolium chloride (INT) assay to assess MDR
reversal potential as described [34]. This initial screening
demonstrated the extract from isolate B158 potentiated (i.e.,
reducing the antibiotic MIC by at least 4-fold) the activity of
erythromycin when tested against E. coli MDR
strains MG1655 ΔBC/pABM, and MG1655 ΔBC/pXYM.
Phylogenetic analysis of 16S rRNA gene sequence indi-
cated that isolate B158 (Genbank Accession no.
MF113387.1) was most closely related to B. denitrificans.
Bioassay-guided fractionation of crude extract generated
Testing of synthetic analogs of 3,6-dihydroxy-1,2-
benzisoxazole
A panel of analogs was synthesized and purchased in order
to probe the structural requirements necessary for the anti-
bacterial effects of 1 as well as to potentially improve
upon its potency (Fig. 1). None of the analogs (2–10)

372
R. W. Deering et al.
Table 1 MIC values for 3,6-dihydroxy-1,2-benzisoxazole (1) against
Gram-negative bacteria in this study
Similarly, antibacterial activity was also completely absent
from the oxazolone analog (10).
Strain
MIC (µg ml−1
)
4-Hydroxybenzoic acid antagonizes the
antibacterial activity of 3,6-dihydroxy-1,2-
benzisoxazole
DM01
MHB
E. coli ATCC 25922TM
0.25-0.5
0.25
8–16
16
>500
64
E. coli UNT 156
P. aeruginosa UCBPP14
>500
500
>500
>500
128
16
We next sought to more stringently test and better under-
stand the mode of action of the active compound. The lower
MICs observed in the minimal medium DM01 versus the
replete growth medium MHB suggested that compounds in
MHB might interfere with the antibiotic activity of 1.
Growth characteristics of A. baumannii Ab197 and
P. aeruginosa UCBPP14 (PA14) were measured using 96-
well phenotype microarray plates (PM1-PM5) purchased
from Biolog, Inc. The bacteria strains were cultured in
DM01 medium both with and without 2 µg ml−1 of com-
pound 1 and growth was measured at 17 and 23 h. These
experiments revealed that 4-hydroxybenzoic acid (4-HB)
and 4-hydroxybenzaldehyde antagonized the antibacterial
effects of 1. To further examine this effect, PA14 was cul-
tured in DM01, both with and without 16 µg ml−1 of 1 and
2-fold serial dilutions of 4-HB and 4-hydroxybenzaldehyde
(Fig. S2). Both 4-hydroxybenzoic acid and 4-
hydroxybenzaldehyde antagonized the growth inhibitory
effects of 1 at the three lowest concentrations tested
(62.5–250 µg ml−1).
P. aeruginosa PA27
K. pneumoniae ATCC 43816TM
K. pneumoniae ATCC BAA-2146TM
A. baumannii UNT 190
>64
1-2
2
A. baumannii UNT 197
2
A. baumannii L1051
nt
12.5
6.25
25
A. baumannii Isolate 9, NR-13382
A. baumannii Naval-81, NR-17786
A. baumannii ATCC 19606TM
nt
nt
nt
50
nt not tested
Molecular docking of 3,6-dihydroxy-1,2-
benzisoxazole onto A. baumannii CPL homology
model
Fig. 1 Panel of compounds tested for MICs against A. baumannii in
SAR study
The phenotypic array highlighted the antagonistic effect
of 4-HB against 1, suggesting our benzisoxazole anti-
biotic may be targeting bacterial metabolic processes that
involve 4-HB (Fig. 2). To examine if compound 1 has the
potential to inhibit chorismate pyruvate-lyase (CPL),
involved in the formation of 4-HB, we performed an in
silico molecular docking study. In the absence of a crystal
structure for A. baumannii CPL, E. coli (UniProt P26602)
CPL was used to create a homology model. Superposition
of the A. baumannii model with E. coli template shows a
root-mean-square distance (RMSD) value of 0.780 Å
(Fig. S3), indicating a good fit to the model. Molecular
docking under the solvated environment finds that com-
pound 1 exhibits a favorable predicted binding affinity of
−5.8 kcal mol−1 (Table S3) and interacts with the primary
site of A. baumannii model (Figs. 3 and S4). The binding
of 4-hydroxybenzoate to E. coli CPL and compound 1 in
A. baumannii involves five hydrogen bonds with four
conserved residues (Arg76/Arg78, Glu155/Glu157, and
peptide amide of Met34/Met35 and Leu114/Leu116)
(Fig. 3).
demonstrated increased potency against the panel of clinical
A. baumannii strains (Table S2). However, structural
requirements for optimal potency were revealed. Replace-
ment of the hydroxyl substituent at C6 (as in 2–6) displayed
pronounced decreases in potency. Based on these data, a
(H-bond) donor substituent at C6 appears to be required for
antibacterial effects, with hydroxyl (1) as the preferred
substituent over amino (5). Though the C6 methoxyl group
(6) did not completely abolish activity, the dramatic
decrease in potency concludes that alkyl modifications to
the C6 hydroxyl group are unlikely to improve potency in
this pharmacophore. Because of the preference of the
hydroxyl substituent at the C6 position, an additional
hydroxyl group was installed at C4 (7), but this abolished
the antibacterial activity.
In addition to substituents on the benzene ring, the
necessity of the isoxazole ring to the pharmacophore was
investigated. Methylation of the nitrogen atom (8 and 9) led
to inactive products; it is noted that this modification leads
to loss of conjugation in the hydroxyl-isoxazole ring.

Identification of a bacteria-produced benzisoxazole with antibiotic activity against multi-drug. . .
373
Fig. 2 Formation and utilization of 4-hydroxybenzoate by bacteria. Conversion of chorismate to 4-hydroxybenzoate (4HB) by chorismate
pyruvate-lyase (CPL) in E. coli, followed by enzymatic synthesis of prenylated-4-hydrobenzoate catalyzed by 4HB octaprenyltransferase
Fig. 3 Binding conformation of 4HB in E. coli CPL (PDB 1FW9) and compound 1 in A. baumannii (GenBank: SST05187.1) model. Hydrogen
bonds (black lines) between ligand (gray) and protein residues (green) are labeled. a Binding of 4HB to E. coli CPL crystal structure (PDB:1FW9).
b Molecular docking of compound 1 onto A. baumannii homology model under solvated (water) environment
Discussion
From the panel of analogs tested, it appears that the
structural features in the natural product 1 are necessary
components of the antibiotic pharmacophore. A hydrogen
bond donor is necessary at C6, and a hydroxyl is preferred
over an amino group. Modifications to the heteroaromatic
ring were found to be detrimental. Further optimization of
the structure for antibacterial effects against A. baumannii
would include determining if the 3-position is modifiable, as
well as exploring other positions on the benzene ring for
substitution.
Our results demonstrate that the antibacterial effect of 1
on A. baumannii can be reversed by supplementing the
growth medium with 4-hydroxybenzoate (4-HB) and, to a
lesser extent, by 4-hydroxybenzaldehyde. 4-HB is a pre-
cursor of ubiquinone biosynthesis for the electron transport
chain and aerobic growth [41–43]. Connecting the antago-
nist effect of 4-HB with possible mechanisms of action, we
have formulated two hypotheses pertaining to two target
enzymes, CPL and 4-HB octaprenyltransferase, both of
which are involved in the production and transformation of
4-HB (Fig. 2).
1,2-benzisoxazoles are bicyclic, heteroaromatic structures
found in many approved drugs, are able to cross the
blood-brain barrier, and have been classified as privileged
structures for drug design for central nervous system
disorders and other therapeutic areas [39, 40]. The het-
erocyclic ring structure of 1 shares similarity with the
clinically used antibiotics cycloserine and linezolid.
Interestingly, cycloserine was inactive against our panel
of A. baumannii strains (data not shown). Oxazolidinone
antibiotics are only effective against Gram-positive
pathogens, and match the ring structure of the inac-
tive 10, further supporting the highly specific nature of the
1,2-benzisoxazole pharmacophore for Gram-negative
pathogens. Compound 1 was previously reported to pos-
sess potent growth inhibition of Gram-negative
bacteria including E. coli, Proteus spp., Salmonella spp.,
K. pneumoniae, Enterobacter spp., and Serratia marces-
cens, as well as low toxicity with an LD50 of over
1500 mg in mice [35, 36]. Previous work indicated no
inhibition was observed with the Gram-positive bacteria
Staphylococcus aureus, Bacillus spp., Micrococcus sp.,
Corynebacterium sp., or the Gram-negative Pseudomonas
spp. [35, 36].
The production of 4-HB differs across animals and bac-
teria. Mammals synthesize 4-HB from tyrosine [41, 43],
while Gram-negative bacteria produce 4-HB from chor-
ismate, a product of the shikimate pathway [41, 44, 45].

374
R. W. Deering et al.
In the first step of ubiquinone biosynthesis, bacterial CPL,
the sole producer of 4-HB [46], irreversibly converts chor-
ismate into 4-HB and pyruvate [46–48] (Fig. 2). The cata-
lytic cycle of bacterial CPL involves a solvent accessible
secondary binding site interconnected to an internal primary
active site [49, 50], where product 4-HB exhibits a ten-fold
higher binding affinity than chorismate [47, 49–51]. This
product inhibition mechanism allows 4-HB to limit ubiqui-
hydrogen to the negatively charged Asp191, which is the-
orized to result in the enhancement of the negative partial
charge at the meta-position and hence activation of the
benzene ring [54] (iii) the necessity of a functional group
that is able to hydrogen bond at the position para to C6 in
order to interact with Arg72 [54]. Both Asp191 and Arg72
are active site residues. Compound 1 satisfies both condition
ii and iii and may mimic 4-HB, hindering prenylation either
by blocking the active site or by getting prenylated itself. In
the former case, ubiquinone cannot be formed without the
prenyl tail. In the latter case, the prenylated products might
not be able to proceed through the ubiquinone biosynthesis
pathway due to the specificity of subsequent enzymes
[56, 57]. A sequence alignment with E. coli’s UbiA amino
acid sequence (Fig. S6) demonstrated that Asp191 is con-
served across three bacteria and two mammalian species,
while Arg72 is conserved across E. coli, A. baumannii,
P. aeruginosa and Mus musculus but not in humans. Hence,
the active site Arg72 could enhance the specificity of 1’s
interaction in bacteria and lower mammals.
The ability to bind to CPL would also account for the
reduced activity of the analogs 2–9. Compounds 2–4, 6, and
9 all lack a C6 hydroxyl group and therefore would not
enable H-bonding from C6 to the active site Asp191.
Compounds 8 and 9 would lack the ability to form an H-
bond from the C3 position to Arg72. With regard to com-
pound 7, it has been previously shown that substitutions to
4-HB at both ortho positions are not accepted as substrates
for 4-HB octaprenyltransferase [55]. Compound 5, which
substitutes an amine in place of the C6 hydroxyl group,
retained modest activity, perhaps because it may still H-
bond with the active site residue Asp191. 4-HB octapre-
nyltransferase has previously been shown to tolerate a
switch from hydroxy to an amino group in the 4-HB sub-
strate [55].
It is interesting that the antibiotic effects of compound 1
are intensified under minimal media conditions. Marine
bacteria frequently compete under minimal nutrient condi-
tions for a good part of their life cycle [58]. Future studies to
decipher the selective pressures that compound 1 are
expressed under could yield more information as to the
natural history of this molecule and allelopathic pathways in
nature.
Narrow spectrum antibacterial drugs are becoming more
desirable due to advances in diagnostic tests and changes to
the regulatory environment [59]. Resistance is expected to
develop more slowly to narrow-spectrum antibiotics and
collateral damage to the gut microbiome, such as antibiotic-
associated colitis, is less likely [60]. Compound 1 was more
active against A. baumannii strains than other Gram-
negative bacteria when measured using standard MIC
assays using Mueller-Hinton broth. Coupled with the cur-
rent paucity of drugs available to treat MDR A. baumannii
none production via
a negative feedback pathway
[49, 51, 52] and stabilize CPL in the absence of chorismate-
binding [50]. When chorismate binds at the secondary site,
this entry encourages the release of 4-HB from the primary
site, where chorismate subsequently enters and is converted
into 4-HB and pyruvate [50]. Pyruvate leaves, while 4-HB
remains bound to CPL for structural stability [50, 51]. The
CPL enzymes of four Gram-negative bacteria (E. coli, A.
baumannii, K. pneumoniae, S. marcescens), which are all
susceptible to 1 [35, 36], share between 83–100% amino
acid identity among the active site residues (Fig. S5), further
bolstering a possible benzisoxazole-mediated binding to
CPL. Furthermore, the lack of CPL in mammals may
account for 1’s low toxicity to mice [35, 36].
Our molecular docking results provide additional support
of the CPL inhibition hypothesis, where compound 1
interacts with the primary site of CPL (Figs. 3 and S4). The
five hydrogen-bonds and four conserved residues observed
in the binding of 4-HB in E. coli are likewise maintained in
compound 1’s interaction with A. baumannii model, thereby
indicating a similar binding conformation. Overall, the
conserved binding interactions suggest that 1 may be a
competitive product inhibitor of CPL, as a sufficient con-
centration of 1 may outcompete 4-HB at the primary
binding site. The absence of 4-HB-binding may disrupt
CPL’s catalytic cycle [50, 51] and prevent the production of
4-HB, thus arresting ubiquinone pathway [46–48] and
aerobic bacterial growth [41, 42].
Following CPL’s production of 4-HB, membrane-bound
4-HB octaprenyltransferase (UbiA) prenylates 4-HB
[41, 47, 48, 53], opening up yet a second speculation for
compound 1’s mechanism of action. 4-HB octaprenyl-
transferase is a membrane-bound enzyme in the ubiquinone
biosynthesis pathway, encoded by the ubiA gene in bacteria
and by the COQ2 gene in mammals [41]. In the first step of
the ubiquinone biosynthesis pathway, the bisubstrate
enzyme binds both 4-HB and geranyl diphosphate to pre-
nylate the meta position of 4-HB to yield 3-polyprenyl-4-
hydroxybenzoate (Fig. 2) [54]. A second hypothesis is then
that 1 competitively inhibits 4-HB binding to UbiA, thereby
preventing ubiquinone biosynthesis. Support for this second
possibility comes from three main conditions: (i) 4-HB
octaprenyltransferase is
a
promiscuous, non-specific
enzyme towards both its substrates [41, 55] (ii) the neces-
sity of a hydrogen bond donor at C6 for transfer of

Identification of a bacteria-produced benzisoxazole with antibiotic activity against multi-drug. . .
375
infections, we suggest that further studies to advance the
development of 1,2-benzisoxazole antibiotics are warranted.
resin was added to the cultures. On the eighth day, the resin
was filtered from the bacterial culture under vacuum,
desalted by rinsing with Milli-Q water, and allowed to dry
overnight at room temperature.
Materials and methods
Metabolites were eluted from the resin first in 100 ml of
(1:1) methanol/dichloromethane, followed by 100 ml of
methanol. This extract was then dried under vacuum
centrifugation (ThermoSavant). Dried extracts were stored
at −80 °C until bacterial susceptibility assays. In sum, 72 l
of B158 culture were processed as described above and
yielded 14.5 g of crude extract. The crude extract was
subjected to silica gel column chromatography with a step
gradient of 100% isooctane, 4:1 isooctane/ethyl acetate;
3:2 isooctane/ethyl acetate, 2:3 isooctane/ethyl acetate, 1:4
isooctane/ethyl acetate, 100% ethyl acetate, 1:1 ethyl
acetate/methanol, and 100% methanol, yielding eight
fractions. Active constituents, as determined with the INT
assay, eluted in 100% ethyl acetate and 1:1 ethyl acetate/
methanol (fractions 6 and 7) were combined (totaling
2374 mg) and further fractionated by SPE/ENVI-18 sep
pak with a step gradient of 19:1 water/acetonitrile, 4:1
water/acetonitrile, 13:7 water/acetonitrile, 1:19 water/
acetonitrile, and 100% acetone. All solvents with the
exception of acetone were acidified with 0.1% formic acid.
Active constituents eluted in 4:1 water/acetonitrile and
13:7 water/acetonitrile. These fractions were combined
(totaling 698.4 mg) and further chromatographed using a
gradient of acetonitrile (0.1% formic acid) and water
(0.1% formic acid) by semipreparative HPLC carried out
on an Agilent 1200 series equipped with an autosampler,
diode array detector, quaternary pump, and 96-well plate
fraction collector with a Phenomenex Luna 5 µm C18(2),
100 Å, LC column (250 × 10 mm) as the stationary phase
with a flow rate of 4 ml min−1. Chromatography methods
were as follows: begin at 5% acetonitrile and ramp to 40%
acetonitrile over 17 min, ramp to 95% acetonitrile over
10 s and hold for 7 min, return to 5% acetonitrile over 10 s
and hold for 4 min at starting conditions. Active con-
stituents eluted between 30–32% acetonitrile with the
resulting activity spread over two wells of a 96-well plate
totaling 32.4 mg. The final purification of active fractions
was achieved by using a gradient of methanol (0.1% for-
mic acid) and water (0.1% formic acid) by analytical
HPLC (Phenomenex Kinetex 2.6 µm C18 100 Å, LC col-
umn (150 × 2.1 mm) as the stationary phase with a flow
rate of 0.2 ml min−1 and UV detection at 276 nm. Chro-
matography methods were as follows: being at 5%
methanol and hold for 5 min, ramp to 40% methanol over
11 min, hold at 40% methanol for 1 min, ramp up to 95%
methanol over 10 s, hold at 95% methanol for 3 min, then
immediately revert to starting conditions and hold for
3 min. Compound 1 (1 mg) eluted at 13.3 min to yield 6-
hydroxy-1,2 benzoxazol-3(2H)-one.
General experimental procedures
NMR experiments were conducted using an Agilent NMRS
500 MHz spectrometer with (CD3)2SO (referenced to resi-
dual DMSO at δH 2.50 and δC 39.5) or CD3OD (referenced
to residual CH3OH at δH 3.31 and δC 49.0) at 25 °C. High
Resolution Electrospray ionization mass spectra (HRE-
SIMS) were acquired using an AB Sciex TripleTOF
4600 spectrometer in the negative ion mode. Flash chro-
matography was completed with a Combiflash Rf200
equipped with a 40 g silica gel RediSepRf High Perfor-
mance Gold column (Teledyne ISCO). HPLC experiments
were performed on a Hitachi Elite LaChrom system
equipped with a diode array detector (DAD) model L-2450,
pump L-2130, and autosampler L-2200. Semi-preparative
HPLC purifications were accomplished with a Waters
XBridge Prep C18 5 µm, 10 × 250 mm column at 4.5 ml
min−1
.
Microbial isolation and identification
A single comb jelly, Mnemiopsis leidyi, was obtained via
collection by dip net at the surface from Eel Pond, Woods
Hole, MA (41° 31’ 32” N, 70° 40’ 12” W). The digestive
contents were stamped onto a tryptone seawater agar (TSW;
1 g l−1 tryptone; 1 l of 75:25 natural seawater:Milli-Q water;
15 g agar) plate and cultivated at 23 °C for three days. A
single semi-transparent colony was transferred onto a fresh
TSW plate to obtain a pure isolate of strain B158. The
isolated strain B158 was identified to be B. denitrificans on
the basis of 97.3% similarity in the 16S rRNA gene
sequence (2370 nucleotides; Genbank Acc. No.
MF113387.1) to B. denitrificans NBRC 105663 (Genbank
Acc. No. AB682257.1). Pure stocks were frozen back in
10% DMSO and stored at −80 °C until use.
Bacteria culture and extract production
A starter culture of B158 was prepared by inoculating 6 ml
of TSW media and with 100 µl of frozen culture and
incubated at 23 °C, 100 rpm for 3 days. Large batch cultures
to scale up growth consisted of 1.5 l of TSY (1 g l−1 tryp-
tone, 1 g l−1 yeast extract, 1 l of 75:25 natural seawater:
Milli-Q water) media inoculated with 1.5 ml of ‘starter’
culture and grown at 100 rpm for 8 days at 23 °C. Twenty-
four hours before culture filtration (day 7), 20 ml of 1:1
mixture of sterile, washed Amberlite XAD-7 and XAD-16

376
R. W. Deering et al.
3,6-dihydroxy-1,2-benzisoxazole (1)
3-hydroxy-6-fluoro-1,2-benzisoxazole (3)
The synthesis shown in Supplementary Fig. 1 is based on
one previously published [61]. Hydroxylamine hydro-
chloride (2.50 g, 36.0 mmol) was added to a stirring solu-
tion of KOH (2.69 g, 47.9 mmol) in water (30 ml) at room
temperature. Methyl 2,4-dihydroxy-benzoate (1.01 g,
6.0 mmol) dissolved in 1,4 dioxane (5 ml) was added
dropwise to the hydroxylamine solution and stirred for 24 h
at room temperature. After removing the 1,4 dioxane in
vacuo, the pH was adjusted to 1 using 3 N HCl and
extracted with ethyl acetate (3 × 50 ml). The organic layers
were combined and dried over Na2SO4, filtered and con-
centrated in vacuo. The resultant residue was purified by
silica gel flash chromatography using a gradient of
20–100% ethyl acetate/hexanes to afford 2,4-dihydroxy-
benzohydroxamic acid (765.7 mg, 76% yield).
Compound 3 was prepared from commercially available
methyl 2-hydroxy-4-fluoro-benzoate according to the
scheme used to prepare 1. 3 was further purified by semi-
preparative HPLC using the method: 12 min gradient from
55–100% MeOH in H2O with 0.1% FA to yield 9.1 mg
1
(white powder). H NMR (500 MHz, CD3OD) δ 7.09 (1H,
td, J = 1.5, 9.1), 7.22 (1H, dd, J = 1.5, 9.1), 7.70 (1H, dd,
J = 5.2, 8.9). 13C NMR (125 MHz, CD3OD) δ 98.3 (d, J =
27.7), 112.8, 113.0 (d, J = 25.7), 123.9 (d, J = 11.5), 165.7
(d, J = 14.4), 166.3 (d, J = 249.0), 167.0. HRMS calcd for
C7H3FNO2: 152.0148. Found [M − H]: 152.0271.
3-hydroxy-6-(benzyloxy)-1,2-benzisoxazole (4)
Commercially available methyl 2,4-dihydroxy-benzoate
(1.0 g, 5.92 mmol) was dissolved in 10 ml acetone and
treated with K2CO3 (1.0 g, 7.24 mmol) and NaI (0.25 g,
1.67 mmol). The resulting suspension was stirred for 10 min
under N2 and then benzyl bromide (700 μl, 5.85 mmol) was
added dropwise. The reaction mixture was stirred for 24 h at
ambient temperature. After removing the acetone in vacuo,
the resulting residue was resuspended in H2O and extracted
with EtOAc (4 × 40 ml). The organic layers were combined,
dried over Na2SO4, and concentrated in vacuo. This residue
(1.8 g) was purified by silica gel flash chromatography
using a gradient of 10–100% EtOAc in hexanes to afford
methyl 2-hydroxy-4-benzyloxy-benzoate (687.4 mg, 45.5%
yield) as white crystals. Methyl 2-hydroxy-4-benzyloxy-
benzoate was then subject to the synthetic steps used to
obtain 1, which afforded 4, which was further purified by
semi-preparative HPLC using an isocratic method of 50%
MeOH in H2O with 0.1% FA. This obtained 11.0 mg (white
2,4-dihydroxy-benzohydroxamic
acid
(161.4 mg,
0.96 mmol) was dissolved in anhydrous tetrahydrofuran
(10 ml) under N2. Dry 1,1′-carbonyldiimidazole (CDI)
(489.2 mg, 3.0 mmol) was added to the mixture and heated
at reflux conditions for 30 min. Et3N (210 μl, 1.5 mmol) was
then added, and the reaction proceeded at reflux for 18 h.
The reaction mixture was then left to cool to room tem-
perature, and THF was removed in vacuo. The resulting
residue was reconstituted in 1 N HCl and extracted with
EtOAc (3 × 50 ml). The combined organic layers were dried
over Na2SO4, filtered and concentrated. The resulting resi-
due was purified by silica gel flash chromatography using a
gradient of 30–50% ethyl acetate with 0.1% formic acid
(FA)/hexanes to afford 3,6-dihydroxy-1,2-benzisoxazole
(107.7 mg, 74% yield, overall yield 56%). Prior to bioassay,
this product was further purified by HPLC with an isocratic
method of 20% MeOH in H2O with 0.1% FA to afford
4.8 mg of 1 (white powder). 1H NMR (500 MHz, CD3OD) δ
6.70 (1H, d, J = 1.9), 6.78 (1H, dd, J = 1.9, 8.7), 7.50 (1H,
d, J = 8.6). 13C NMR (125 MHz, CD3OD) δ 96.2, 108.2,
114.5, 123.6, 163.2, 166.9, 167.6. HRMS calcd for
C7H4NO3: 150.0191. Found [M − H] 150.0206.
1
powder). H NMR (500 MHz, (CD3)2SO) δ 5.17 (2H, s),
6.94 (1H, d, J = 8.7), 7.15 (1H, s), 7.34 (1H, t, J = 7.4),
7.40 (2H, t, J = 7.4), 7.46 (2H, d, J = 7.4), 7.60 (1H, d, J =
8.6). 13C NMR (125 MHz, (CD3)2SO) δ 69.9, 94.5, 108.3,
113.4, 122.0, 127.9 (2 C), 128.1, 128.5 (2 C), 136.5, 161.1,
164.9, 165.7. HRMS calcd for C14H10NO3: 240.0661.
Found [M − H] 240.0564.
3-hydroxy-1,2-benzisoxazole (2)
Compound 2 was prepared from commercially available
methyl salicylate (methyl 2-hydroxy-benzoate) according to
the scheme used to prepare 1. 2 was further purified by
semi-preparative HPLC using an isocratic method of 50%
MeOH in H2O with 0.1% FA to afford 6.0 mg of 2 (white
powder). 1H NMR (500 MHz, (CD3)2SO) δ 7.31 (1H, t, J =
7.0), 7.54 (1H, d, J = 8.2), 7.59 (1H, t, J = 7.9), 7.75
(1H, d, J = 8.0), 12.45 (1H, br s). 13C NMR (125 MHz,
3-hydroxy-6-amino-1,2-benzisoxazole (5)
Compound 5 was prepared from commercially available
methyl 2-hydroxy-4-amino-benzoate according to the
scheme used to prepare 1 with minor modification. The
ring-closing step was accomplished with CDI/MeCN
instead of CDI/THF/Et3N as used for 1. The resulting
mixture was purified using semi-preparative HPLC with a
gradient method from 10–55% MeOH in H2O with 0.1%
(CD3)2SO)
δ
110.0, 114.6, 121.4, 122.9, 130.5,
1
163.0, 165.4. HRMS calcd for C7H4NO2: 134.0242. Found
FA. This afforded 1.1 mg of 5 (white powder). H NMR
[M − H] 134.0251.
(500 MHz, (CD3)2SO) δ 5.80 (2H, s), 6.39 (1H, d, J = 1.4),

Identification of a bacteria-produced benzisoxazole with antibiotic activity against multi-drug. . .
377
6.50, (1H, dd, J = 1.5, 8.4), 7.30 (1H, d, J = 8.4). 13C NMR
(125 MHz, (CD3)2SO) δ 91.2, 103.6, 111.8, 121.8, 152.3,
165.6, 165.9. HRMS calcd for C7H5N2O2: 149.0351. Found
[M − H] 149.0323.
6-hydroxy-2-benzoxazolinone (10)
Compound 10 was purchased commercially (abcr GmbH).
Biological assays
3-hydroxy-6-methoxy-1,2-benzisoxazole (6)
Media Luria Bertani (LB) medium and cation-adjusted
Mueller-Hinton broth (MHB) were purchased from Sigma-
Aldrich. DM01 medium contains 25 mM potassium phos-
phate, pH 6 (3.3 mM K2HPO4, 21.7 mM KH2PO4), 1 mM
MgSO4, 100 µM CaCl2, 75 mM sodium pyruvate, 1 g l−1
NH4Cl.
Compound
Aldrich).
6 was purchased commercially (Sigma–
3,4,6-trihydroxy-1,2-benzisoxazole (7)
Compound 7 was prepared from commercially available
methyl 2,4,6-trihydroxy-benzoate according to the scheme
used to prepare 1. 7 was further purified from a mixture by
Bacterial susceptibility determinations Antimicrobial
activity of marine bacterial extracts, semi-purified fractions,
and pure compounds were evaluated by whole-cell assays
with E. coli isolates expressing one of three archetype RND
efflux pumps (AcrAB-TolC, MexAB-OprM, and MexXY-
OprM) that are known to contribute to antibiotic resistance
in Enterobacteriaceae and P. aeruginosa clinical isolates.
Each of the three E. coli isolates overexpressing RND efflux
pumps were used in 96-well plate-based assays to determine
bacterial susceptibility of B158 crude extract or fraction
tested in duplicate at 1 mg ml−1 and determined by
rapid p-iodonitrotetrazolium chloride colorimetric assay as
described in [34]. Minimum inhibitory concentrations
(MICs; defined as the lowest concentration that results in no
visible growth) of the synthetically derived compound 1
was determined using a 2-fold standard microdilution
method in Muller-Hinton broth (MHB) in microtiter plates
as defined by the National Committee for Clinical Labora-
tory Standards [62] for each of the three RND over-
expressing E. coli strains.
semi-preparative HPLC with
a gradient method of
25–100% MeOH in H2O with 0.1% FA. This afforded
1
6.8 mg of 7 (white powder). H NMR (500 MHz, (CD3)
2SO) δ 6.08 (1H, d, J = 1.3), 6.14 (1H, d, J = 1.3), 10.22
(3H, br s). 13C NMR (125 MHz, (CD3)2SO) δ 86.8, 96.6,
97.3, 153.8, 161.9, 165.2, 166.6. HRMS calcd for
C7H4NO4: 166.0140. Found [M − H] 166.0151.
N-methyl-6-hydroxy-1,2-benzisoxazol-3(2H)-one (8)
and N-methyl-6-methoxy-1,2-benzisoxazol-3(2H)-
one (9)
Compounds 8 and 9 were prepared from 1 in a one-step
synthesis and purification. 1 (46 mg, 0.33 mmol) was dis-
solved in dry MeCN (4 ml), and K2CO3 (91 mg, 0.66 mmol)
was suspended in the solution. Suspension was stirred
rapidly under N2, and excess CH3I (260 μl, 4.2 mmol) was
added dropwise and stirred for 18 h at room temperature.
The reaction was quenched by removing MeCN in vacuo,
and the resulting residue was dissolved in 1 N HCl. This
solution was extracted with EtOAc (3 × 30 ml), and the
combined organic layers were dried over Na2SO4, filtered
and concentrated in vacuo. The resulting products were
purified by semi-preparative HPLC using a 12 min gradient
from 35–80% MeOH in H2O with 0.1% FA to afford 8
(12.1 mg, white powder) and 9 (8.5 mg, white powder).
Pathogen strains and MIC testing A. baumannii UNT190-
1, A. baumannii UNT197-1, and E. coli UNT156-1 are
clinical isolates kindly provided by Dr. William Weiss at
North Texas University. P. aeruginosa strain UCBPP-PA14
was kindly provided by Dr. Frederick M. Ausubel. E. coli
EC2 ((Migula) Castellani and Chalmers; ATCC 25922TM
,
,
K. pneumoniae ATCC 43816TM and ATCC BAA-2146TM
1
Compound 8 H NMR (500 MHz, (CD3)2SO) δ 3.46
and A. baumannii ATCC 19606TM were obtained from the
American Type Culture Collection. A. baumannii L1051
was obtained from a patient skin sample at Rhode Island
Hospital (Providence, RI) and is maintained in the LaPlante
laboratory. A. baumannii, Isolate 9, NR-13382 and A.
baumannii, Strain Naval-81, NR-17786 are human blood
isolates obtained from the Biodefense and Emerging
Infection Research Resources Repository (BEI) Resources,
NIAID, NIH. L1051 and NR-13382 are considered multi-
drug resistant organisms (MDROs; non-susceptibility to one
agent in three antimicrobial classes), according to pre-
viously described definitions [37, 38]. Minimum inhibitory
(3H, s), 6.68 (1H, d, J = 1.6), 6.57 (1H, dd, J = 1.6, 8.4),
7.55 (1H, d, J = 8.4), 10.73 (1H, br s). 13C NMR (125 MHz,
(CD3)2SO) δ 33.3, 95.3, 106.8, 113.4, 124.7, 162.3, 163.4,
164.2. HRMS calcd for C8H8NO3: 166.0504. Found [M +
H]: 166.0216.
1
Compound 9 H NMR (500 MHz, (CD3)2SO) δ 3.51
(3H, s), 3.86 (3H, s), 6.89 (1H, dd, J = 2.1, 8.6), 7.05 (1H,
d, J = 2.0), 7.64 (1H, d, J = 8.6). 13C NMR (125 MHz,
(CD3)2SO) δ 33.2, 56.1, 93.8, 108.1, 113.2, 124.5, 162.2,
163.7, 164.5. HRMS calcd for C9H10NO3: 180.0661. Found
[M + H]: 180.0341.

378
R. W. Deering et al.
concentrations (MICs) were determined in duplicate by
broth microdilution in accordance with Clinical Laboratory
Standards Institute (CLSI) standards [63, 64].
[70, 71]. A grid box was used to define the search parameter
[70, 71]. Ten protein-ligand models were produced and
ranked by docking scores. The most negatively scored
protein-ligand model containing >3 H-bonds was selected.
H-bonds and Van der Waals interactions were predicted
using FindHBond and Find Clashes/Contacts function of
UCSF Chimera [70, 72].
Phenotype microarray experiments Five different types of
96-well phenotype microarray plates (PM1-PM5) were
purchased from Biolog, Inc. A 2 ml overnight bacteria
culture (A. baumannii strain UNT197 or P. aeruginosa
UCBPP14) in LB was grown in a shaking incubator at
37 °C. The culture was pelleted by centrifugation, washed,
and resuspended in the same volume of phosphate-buffered
saline (PBS). DM01 medium with or without 2 µg ml−1 of 1
were each inoculated with 1/1000 volume of the resus-
pended culture. 100 µl of the inoculated medium was added
to each well of the phenotype microarray plates. Plates were
covered and incubated at 37 °C. Plates were transferred to a
Spectramax i3x plate reader (Molecular Devices) for mea-
surement of OD600 after 17 and 23 h.
Acknowledgements RD was financially supported by the Omar
Magnate Foundation Fellowship. Research reported in this publication
was made possible by the use of equipment and services available
through the RI-INBRE Centralized Research Core Facility, which is
supported by the Institutional Development Award (IDeA) Network
for Biomedical Research Excellence from the National Institute of
General Medical Sciences of the National Institutes of Health under
grant number P20GM103430. Additional financial support to KEW
and TJM was provided by the National Institute of Allergy and
Infectious Disease of the National Institutes of Health under grant
number 1R21AI119311-01. Acinetobacter baumannii, Isolate 9, NR-
13382 and Acinetobacter baumannii, Strain Naval-81, NR-17786 were
obtained through BEI Resources, NIAID, NIH.
Targeted antibiotic antagonism experiments (P. aeruginosa
UCBPP14) A 2 ml overnight culture of P. aeruginosa
UCBPP14 in LB was grown in a shaking incubator at
37 °C. The culture was pelleted by centrifugation, washed,
and resuspended in the same volume of phosphate-buffered
saline (PBS). DM01 medium was supplemented with 2-fold
serial dilutions of 4-hydroxybenzoic acid or 4-
hydroxybenzaldehyde (Sigma-Aldrich) both with and
without 16 µg ml−1 of 1, then inoculated with 1/1000
volume of the bacteria suspension. The cultures were
incubated at 37 °C, and OD600 was measured at 17 h.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note Springer Nature remains neutral with regard to
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Open Access This article is licensed under a Creative Commons
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long as you give appropriate credit to the original author(s) and the
source, provide a link to the Creative Commons license, and indicate if
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article are included in the article’s Creative Commons license, unless
indicated otherwise in a credit line to the material. If material is not
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use is not permitted by statutory regulation or exceeds the permitted
use, you will need to obtain permission directly from the copyright
holder. To view a copy of this license, visit http://creativecommons.
org/licenses/by/4.0/.
Sequence alignment and homology modeling of CPL The
amino acid sequence of A. baumannii chorismate pyruvate-
lyase (CPL) (GenBank: SST05187.1) was aligned with
template E. coli CPL (PDB 1FW9), which has 85% query
cover, E-value of 3 ×10−41, and 50.35% identity. Sequence
alignment was done using MUSCLE [65]. The solvent-free
A. baumannii homology model was created by Modeller
9.24 and refined using Modeller 9.2’s salign_2d [66, 67].
Homology model with GA341 value of 1.00 and the most
negative DOPE was selected [66]. The homology model
was quality assessed with PROCHECK [68]. To evaluate
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