Discovery of antibiotic (E)-3-(3-carboxyphenyl)-2-(4-cyanostyryl)quinazolin-4(3 H)-one

Article abstract of DOI:10.1021/jacs.5b00056

In the face of the clinical challenge posed by resistant bacteria, the present needs for novel classes of antibiotics are genuine. In silico docking and screening, followed by chemical synthesis of a library of quinazolinones, led to the discovery of (E)-3-(3-carboxyphenyl)-2-(4-cyanostyryl)quinazolin-4(3H)-one (compound 2) as an antibiotic effective in vivo against methicillin-resistant Staphylococcus aureus (MRSA). This antibiotic impairs cell-wall biosynthesis as documented by functional assays, showing binding of 2 to penicillin-binding protein (PBP) 2a. We document that the antibiotic also inhibits PBP1 of S. aureus, indicating a broad targeting of structurally similar PBPs by this antibiotic. This class of antibiotics holds promise in fighting MRSA infections.

Full text of DOI:10.1021/jacs.5b00056

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Communication
Discovery of Antibiotic (E)-3-(3-Carboxyphenyl)-2-
(4-cyanostyryl)quinazolin-4(3H)-one
Renee Bouley, Malika Kumarasiri, Zhihong Peng, Lisandro H. Otero, Wei Song, Mark A
Suckow, Valerie A. Schroeder, William R. Wolter, Elena Lastochkin, Nuno T. Antunes,
Hualiang Pi, Sergei Vakulenko, Juan A. Hermoso, Mayland Chang, and Shahriar Mobashery
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b00056 • Publication Date (Web): 28 Jan 2015
Downloaded from http://pubs.acs.org on January 31, 2015
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Discovery of Antibiotic (E)-3-(3-Carboxyphenyl)-2-(4-
cyanostyryl)quinazolin-4(3H)-one
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Renee Bouley†, Malika Kumarasiri†, Zhihong Peng†, Lisandro H. Otero‡, Wei Song†, Mark A. Suckow§,
Valerie A. Schroeder§, William R. Wolter§, Elena Lastochkin†, Nuno T. Antunes†, Hualiang Pi†, Sergei
Vakulenko†, Juan A. Hermoso‡, Mayland Chang*†, Shahriar Mobashery*†
†Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556. ‡Department of Crystallography and
Structural Biology, Instituto de QuímicaꢀFísica “Rocasolano”, Consejo Superior de Investigaciones Científicas, Madrid,
Spain. §Freimann Life Sciences Center and Department of Biological Sciences, University of Notre Dame, Notre Dame, Inꢀ
diana 46556.
Supporting Information Placeholder
terial activity against Escherichia coli and the ESKAPE panel of
bacteria, comprised of Enterococcus faecium, Staphylococcus
aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseuꢀ
domonas aeruginosa, and Enterobacter species, which account
for the majority of nosocomial infections.1,2,6 Antibiotic 1 was
discovered in this effort, with a minimalꢀinhibitory concentration
(MIC) of 2 ꢁg/mL against S. aureus ATCC 29213 of the ESKAPE
panel. The compound also had modest activity against E. faecium
(MIC of 16 μg/mL), but did not have activity against Gramꢀ
negative bacteria of our panel. We initiated lead optimization of
this structural template to improve its in vitro potency, while imꢀ
parting in vivo properties. We synthesized 80 analogs of comꢀ
pound 1 and screened them for in vitro antibacterial activity, metꢀ
abolic stability, in vitro toxicity, efficacy in an in vivo mouse
MRSA infection model, and pharmacokinetics (PK).
ABSTRACT: In the face of the clinical challenge by resistant
bacteria, the present needs for novel classes of antibiotics are
genuine. In silico docking and screening, followed by chemical
synthesis of a library of quinazolinones, led to the discovery of
(E)ꢀ3ꢀ(3ꢀcarboxyphenyl)ꢀ2ꢀ(4ꢀcyanostyryl)quinazolinꢀ4(3H)ꢀone
(compound 2) as an antibiotic effective in vivo against methicillinꢀ
resistant Staphylococcus aureus (MRSA). This antibiotic impairs
cellꢀwall biosynthesis as documented by functional assays, showꢀ
ing binding of 2 to penicillinꢀbinding protein (PBP) 2a. We docꢀ
ument that the antibiotic also inhibits PBP1 of S. aureus, indicatꢀ
ing a broad targeting of structurally similar PBPs by this antibiꢀ
otic. This class of antibiotics holds promise in fighting MRSA
infections.
Emergence of resistance to antibiotics over the past few decades
has created a state of crisis in treatment of bacterial infections.1,2
Over the years, βꢀlactams were antibiotics of choice for treatment
of S. aureus infections. However, these agents faced obsolescence
with the emergence of methicillinꢀresistant S. aureus (MRSA).
Presently, vancomycin, daptomycin, linezolid, or ceftaroline are
used for treatment of MRSA infections, although only linezolid
can be dosed orally. Resistance to all four has emerged. Thus,
new antiꢀMRSA antibiotics are sought, especially agents that are
orally bioavailable. We disclose in this report a new antibiotic
(E)ꢀ3ꢀ(3ꢀcarboxyphenyl)ꢀ2ꢀ(4ꢀcyanostyryl)quinazolinꢀ4(3H)ꢀone,
with potent activity against S. aureus, including MRSA. We docꢀ
ument that quinazolinones of our design are inhibitors of cellꢀwall
biosynthesis in S. aureus, and do so by binding to ᴅᴅꢀ
transpeptidases involved in crosslinking of the cell wall. We reꢀ
port that quinazolinones possess activity in vivo and are orally
bioavailable. This antibiotic holds promise in treating difficult
infections by MRSA.
We used the Xꢀray structure of PBP2a3 to computationally
screen 1.2 million drugꢀlike compounds from the ZINC database4
for binding to the active site using crossꢀdocking with multiple
scoring functions. Starting with highꢀthroughput virtual screening,
the filtering was stepwise with increasing stringency, such that at
each stage the best scoring compounds were fed into the next
stage. The final docking and scoring step involved Glide5 refineꢀ
ment of docking poses with the extra precision mode, where the
top 2,500 poses were clustered according to structural similarity.
Of these, 118 high rankers were purchased and tested for antibacꢀ
Antibiotic 2 emerged from these studies with the desired attribꢀ
utes, including efficacy in a mouse infection model. Antibiotic 2
was synthesized using a variation of a previously reported method
for construction of the quinazolinone core (Scheme 1).7,8 This
synthesis uses anthranilic acid (3) as a precursor, which is cyꢀ
clized to the 2ꢀmethylbenzoxazinone intermediate (4) using reꢀ
fluxing orthoꢀtriethylacetate, in 72% yield. The intermediate is
then subjected to ringꢀopening and ringꢀclosing amidation with
the corresponding aniline derivative in refluxing acetic acid to
give the 2ꢀmethylquinazolinone intermediate (5) with a yield of
92%. The final reaction is an aldolꢀtype condensation with the
respective aromatic aldehyde to give the 2ꢀstyrylquinazolinone
product in 85% yield. Antibiotic 2 showed activity against MRSA
strains similar to that of linezolid and vancomycin. Furthermore,
activity was documented against vancomycinꢀ and linezolidꢀ
resistant MRSA strains (Table 1). In the XTT cell proliferation
assay using HepG2 cells, antibiotic 2 had an IC50 of 63 ± 1 ꢁg/mL,
a value well above the concentration range that manifested the
antibacterial activity. We also showed that the antibiotic was not
an alkylating agent (Figure S1), it did not form glutathione adꢀ
ducts, and showed no hemolysis (<1%) of red blood cells at 50
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Table 1. A comparison of the in vitro antibacterial activity of antibiotic 2 to marketed antibiotics against a panel of Staphy-
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2
3
4
5
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8
lococcal strains.
vancomycin
linezolid
oxacillin
2
S. aureus ATCC 29213a
S. aureus NRS128b
S. aureus NRS70c
2
4
1
1
4
1
0.25
0.5
2
1
1
32
S. aureus NRS123d
S. aureus NRS100e
S. aureus NRS119f
2
1
2
32
16
8
2
2
512
512
512
512
256
128
0.25
2
32
32
2
9
S. aureus NRS120f
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2
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S. aureus VRS1g
S. aureus VRS2h
16
2
512
64
16
2
2
S. epidermidis ATCC 35547
S. haemolyticus ATCC 29970
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1
1
2
a
In vitro activity reported as minimalꢀinhibitory concentration (MIC) in ꢁg/mL. Quality control methicillinꢀsensitive S. aureus (MSSA)
b
c
strain; MSSA strain, mecA negative, resistant to erythromycin, clindamycin, and penicillin; Clinical MRSA strain isolated in Japan,
d
mecA positive, resistant to erythromycin, clindamycin, oxacillin, and penicillin; Communityꢀacquired MRSA strain, mecA positive,
resistant to methicillin, oxacillin, penicillin, and tetracycline; eMRSA strain, mecA positive, resistant to oxacillin, penicillin, and tetracyꢀ
cline; fClinical MRSA strain, mecA positive, resistant to linezolid, ciprofloxacin, gentamicin, oxacillin, penicillin, and trimeꢀ
thoprim/sulfamethoxazole; gClinical MRSA isolate from Michigan, mecA positive, vanA positive, resistant to vancomycin, ciprofloxacin,
clindamycin, erythromycin, gentamicin, oxacillin, and penicillin; hClinical MRSA isolate from Pennsylvania, mecA positive, vanA posiꢀ
tive, resistant to vancomycin, ciprofloxacin, clindamycin, erythromycin, gentamicin, oxacillin, and penicillin.
ꢁg/mL, indicating that the compound was not toxic at concentraꢀ
tions in which antibacterial activity was documented. Furtherꢀ
more, antibiotic 2 was stable in mouse plasma (halfꢀlife of 141 h),
and was metabolically stable in rat and human S9 (100% of 2
remaining after 1ꢀh incubation), liver fractions containing microꢀ
somes (including cytochrome P450 enzymes capable of phase I
metabolism) and cytosol (containing transferases capable of phase
II metabolism). In addition, human plasma protein binding was
determined to be 96.5 ± 0.7%. Antibiotic 2 in its sodium salt form
is water soluble, with a high solubility of 8 mg/mL.
or cell wall (peptidoglycan), respectively. Inhibition of radioꢀ
labeled precursor incorporation by antibiotic 2 at a concentration
of 0.5 MIC was compared with those of known inhibitors of each
pathway (ciprofloxacin, rifampicin, tetracycline, and fosfomyꢀ
cin/meropenem, respectively). As per our design paradigm, antiꢀ
biotic 2 showed notable inhibition of cellꢀwall biosynthesis in
these assays (51 ± 12% compared to 64 ± 8% for fosfomycin and
61 ± 4% compared to 64 ± 2% for meropenem) and did not signifꢀ
icantly affect replication, transcription, or translation (Figures 1,
S4 and S5). To further validate these results, additional in vitro
transcription and translation assays were performed using a T7
transcription kit and an E. coli S30 extract coupled with a βꢀ
galactosidase assay system, respectively. Antibiotic 2 did not
show inhibition of either transcription or translation using these
assays (Figure S6).
Scheme 1. Synthesis of quinazolinone 2
100
Compound 2
Positive control
80
60
40
20
0
Quinazolinone 2 demonstrated excellent in vivo efficacy in the
mouse peritonitis model of MRSA infection,9 with a median efꢀ
fective dose (ED50, the dose that results in survival of 50% of the
animals) of 9.4 mg/kg after intravenous (iv) administration (Figꢀ
ure S2). After a single 10 mg/kg iv dose of 2, plasma levels of 2
were sustained above MIC for 2 h and declined slowly to 0.142 ±
0.053 ꢁg/mL at 24 h (Figure S3). The compound had a volume of
distribution of 0.3 L/kg (Table S1), a long elimination halfꢀlife
(>20 h), and low clearance of 6.87 mL/min/kg, less than 10% of
hepatic blood flow in mice. After a single 10 mg/kg oral (po) dose
of 2, a maximum concentration of 1.29 ꢁg/mL was achieved at 1
h. The terminal halfꢀlife was long (>20 h) and the absolute oral
bioavailability was 50% (Table S1).
DNA
RNA
Protein
Peptidogylcan
Figure 1. Macromolecular synthesis assays. Antibiotic 2 at ½
the MIC. Positive controls for DNA, RNA, protein, and peptiꢀ
doglycan synthesis are ciprofloxacin (0.5 ꢁg/mL), rifampicin (8
ng/mL), tetracycline (31 ng/mL), and fosfomycin (16 ꢁg/mL),
respectively. The maximum inhibition observed for antibiotic 2 is
reported at 120 min of incubation in all cases, except for DNA
synthesis, which was at 80 min.
The mode of action of 2 was investigated by macromolecular
synthesis assays with S. aureus ATCC 29213 (an MSSA strain) in
the logarithmic phase,10 which monitor incorporation of radioꢀ
labeled precursors [methylꢀ3H]ꢀthymidine, [5,6ꢀ3H]ꢀuridine, ʟꢀ
[4,5ꢀ3H]ꢀleucine, or ᴅꢀ[2,3ꢀ3H]ꢀalanine into DNA, RNA, protein,
Having demonstrated that biosynthesis of cell wall is attenuated
by antibiotic 2, we next explored if it would inhibit purified reꢀ
combinant PBP2a by a competition assay with Bocillin FL, a cellꢀ
wall mimetic fluorescent penicillin reporter reagent.11,12 This inꢀ
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hibition assay for PBPs has the limitation in that Bocillin FL is a
spatial positions of certain residues (Lys406, Lys597, Ser598,
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5
6
7
8
covalent modifier of the active site of PBPs and the equilibrium is
inexorably in favor of the irreversible acylation of the activeꢀsite
serine by the reporter molecule. As such, the degree of inhibition
by the nonꢀcovalent inhibitor (e.g., antibiotic 2) will be underesꢀ
timated. Antibiotic 2 was able to inhibit Bocillin FL labeling of
the active site of PBP2a in a competitive and doseꢀdependent
manner, with an apparent IC50 of 140 ± 24 ꢁg/mL (Figures 2A and
S7A). It is important to note that we have observed antibiotic
activity for quinazolinone 2 in strains of S. aureus that do not
express PBP2a (Table 1), which indicated that the antibiotic is
likely to bind to other PBPs as well. This is akin to the case of βꢀ
lactam antibiotics, which bind to multiple PBPs due to high strucꢀ
tural similarity at the active sites. To demonstrate the ability to
bind to other PBPs, membrane preparations of S. aureus ATCC
29213 (the MSSA strain used in the macromolecular synthesis
assays) were used to assess broader PBP inhibition by antibiotic 2.
Inhibition of PBP1 was observed, with an apparent IC50 of 78 ± 23
ꢁg/mL (Figures 2B and S7B). Inhibition of PBP1 of S. aureus
accounts for the antibacterial activity of imipenem and meroꢀ
penem, two carbapenem antibiotics, in MSSA strains.13,14 Howevꢀ
er, both antibiotics are ineffective against MRSA. Because of the
lowꢀcopy numbers of PBP2a in the membranes from MRSA, we
could not demonstrate PBP2a inhibition in the membrane preparaꢀ
tions directly.
Glu602, and Met641) within the active site of the complex, which
could be due to interactions with an antibiotic molecule, but denꢀ
sity for it was not observed. The competitive mode of inhibition
of Bocillin FL binding to the active site by antibiotic 2 was docuꢀ
mented above and is consistent with this scenario. However, some
of these alternative activeꢀsite conformations could also have
come about due to the allosteric triggering. Nevertheless, the antiꢀ
biotic binding at the allosteric site was unanticipated. Determinaꢀ
tion of the binding affinity at the allosteric site was performed
using intrinsic fluorescence quenching of purified PBP2a, which
had been modified covalently within the active site by the antibiꢀ
otic oxacillin.17 Hence, compound 2 would be expected to bind
only to the allosteric site. A Kd of 6.9 ± 2.0 ꢁg/mL was determined
(Figure 4).
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Figure 2. Inhibition of Bocillin FL binding to S. aureus PBP2a
and PBP1. (A) Fluorescence labeling of recombinant purified
PBP2a (1 ꢁM) by Bocillin FL (20 ꢁM) in the presence of increasꢀ
ing amounts of antibiotic 2 (ꢁg/mL). (B) Fluorescence labeling of
S. aureus membrane preparation (150 ꢁg) by Bocillin FL (30 ꢁM)
in the presence of increasing amounts of antibiotic 2 (ꢁg/mL). For
data fitting, consult Figure S7.
Targeting of PBP1 and PBP2a by antibiotic 2 was further conꢀ
firmed by the antiꢀsense methodology developed by Merck workꢀ
ers.15 MRSA COL was transformed individually with plasmids
bearing antiꢀsense fragments for pbpA (encoding PBP1) and
pbp2a (encoding PBP2a) under a xyloseꢀinducible promoter. The
transcribed antiꢀsense fragment under xylose induction binds to
the complementary mRNA for the corresponding PBP, which
leads to diminution of translation, hence hyperꢀsensitizing the
strain to the antibiotic that targets that given PBP. Attenuation of
expression of either PBP1 or PBP2a by this technology sensitized
the strain to antibiotic 2, as it did to imipenem and to ceftaroline,
known PBP inhibitors (Figure S8).
Figure 3. Crystal structure of S. aureus PBP2a in complex with
quinazolinone 2. (A) Ribbon representation of PBP2a showing
antibiotic 2 (in cyan for carbons) bound to the allosteric site. The
allosteric domain spans residues 27ꢀ326, where the Nꢀterminal
domain (residues 27ꢀ138) is shown in green and the remaining
allosteric domain is colored yellow. The transpeptidase domain
(residues 327ꢀ668) is shown in purple. (B) Ribbon superimposiꢀ
tion at the active site of apo PBP2a (PDB ID: 1VQQ) colored in
orange and complex colored in purple. Structural changes occur at
loops α9ꢀβ3, β3ꢀβ4, and β5ꢀα10. (C) Key interactions of antibiotic
2 at the allosteric site. Saltꢀbridge interactions with K273 and
K316 are shown as black dashed lines, the distances are 2.9 Å. Piꢀ
stacking interactions are observed with Y105 and Y297. Stereoꢀ
view showing the refined electron density (2FoꢀFc featureꢀ
enhanced map) for the ligand (blue mesh) contoured at 1.0 σ.
We sought to determine the Xꢀray structure for the complex of
quinazolinone 2 and PBP2a. Soaking experiments of PBP2a crysꢀ
tals with antibiotic 2 resulted in a highꢀresolution structure at 1.95
Å of resolution for the complex (Table S2). This structure reꢀ
vealed density for antibiotic 2 bound to the allosteric site of
PBP2a at 60ꢀÅ distance from the ᴅᴅꢀtranspeptidase active site
(Figure 3). The allosteric site of PBP2a has recently been deꢀ
scribed.16 The critical binding of ligands such as the nascent cellꢀ
wall peptidoglycan at the allosteric site leads to the opening of the
active site, enabling catalysis by PBP2a. The structure revealed
both alteration of three loops surrounding the activeꢀsite pocket,
α9ꢀβ3, β3ꢀβ4, and β5ꢀα10 loops (Figure 3B), and changes in the
Our data can be interpreted as binding of antibiotic 2 both at the
active site (competitive inhibition) and at the allosteric site of
PBP2a or as binding at the allosteric site with negative allosteric
inhibition of the active site of PBP2a. Inhibition of PBP1 is at the
active site, as it lacks allosteric regulation. The importance of both
PBPs to mechanism of 2 was validated by the antiꢀsense technolꢀ
ogy. We further provide Xꢀray crystallography evidence that the
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antibiotic binds to the allosteric site of PBP2a. These data support
material is available free of charge via the Internet at
http://pubs.acs.org.
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4
PBP inhibition by 2, but they do not exclude the possibility for a
pleiotropic effect that could result in inhibition of one or more
additional target(s) in S. aureus. However, if the pleiotropic effect
exists, it manifests itself in another step of the biosynthesis of cell
wall based on the macromolecular synthesis assay results.
AUTHOR INFORMATION
Corresponding Authors
5
mchang@nd.edu or mobashery@nd.edu
6
7
8
Notes
The authors declare no competing financial interests.
9
ACKNOWLEDGMENT
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R.B. was supported by training grant T32GM075762 and by an
individual Ruth L. Kirschstein National Research Service Award
F31AI115851 from the National Institutes of Health and by an
American Chemical Society Division of Medicinal Chemistry
Predoctoral Fellowship. The research in Spain was supported by
grants BFU2011ꢀ25326 and S2010/BMDꢀ2457. The antiꢀsense
strains were generous gifts from Dr. Terry Roemer of Merck.
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(18)Fisher, J. F.; Meroueh, S. O.; Mobashery, S. Chem. Rev. 2005,
105, 395.
(19)Llarrull, L. I.; Testero, S. A.; Fisher, J. F.; Mobashery, S. Curr.
Opin. Microbiol. 2010, 13, 551.
Figure 4. Binding of quinazolinone 2 to PBP2a allosteric site,
determined using a previously described procedure.17 (A) The
change in the maximum fluorescence intensity gave a Kd of 6.9 ±
2 ꢁg/mL (average of three experiments; nonlinear regression for
data fitting with an R2 of 0.9997). (B) Emission scans of PBP2a
intrinsic fluorescence with excitation at 280 nm. Antibiotic 2 was
titrated in to give the final concentrations shown.
In summary, we have described the discovery of a new antibiotic
that exhibits in vitro and in vivo activity against S. aureus, and its
problematic kin MRSA and its resistant variants to other antibiotꢀ
ics. βꢀLactam antibiotics are known inhibitors of PBPs, which are
essential enzymes in cellꢀwall biosynthesis. Resistance to βꢀ
lactam antibiotics is widespread among pathogens, and in the case
of MRSA, it encompasses essentially all commercially available
drugs.18,19 In light of the fact that quinazolinones are nonꢀβꢀlactam
in nature, they circumvent the known mechanisms of resistance to
βꢀlactam antibiotics. As such, quinazolinones hold great promise
in recourse against MRSA as a clinical scourge that kills approxꢀ
imately 20,000 individuals annually in the US alone.20
ASSOCIATED CONTENT
Supporting Information
Experimental details, in vitro toxicity, in vivo efficacy, PK data, in
vitro transcription and translation assay results, PBP2a allosteric
site assay data, MRSA antiꢀsense results, xꢀray crystallographic
statistics, and regression curves. The crystallographic coordinates
are deposited in the Protein Data Bank (PDB code 4CJN). This
(20)Fishbach, M. A.; Walsh, C. T. Science 2009, 325, 1089.
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