Antibiotic That Inhibits the ATPase Activity of an ATP-Binding Cassette Transporter by Binding to a Remote Extracellular Site

Article abstract of DOI:10.1021/jacs.7b04726

Antibiotic-resistant strains of Staphylococcus aureus pose a major threat to human health and there is an ongoing need for new antibiotics to treat resistant infections. In a high throughput screen (HTS) of 230 000 small molecules designed to identify bioactive wall teichoic acid (WTA) inhibitors, we identified one hit, which was expanded through chemical synthesis into a small panel of potent compounds. We showed that these compounds target TarG, the transmembrane component of the two-component ATP-binding cassette (ABC) transporter TarGH, which exports WTA precursors to the cell surface for attachment to peptidoglycan. We purified, for the first time, a WTA transporter and have reconstituted ATPase activity in proteoliposomes. We showed that this new compound series inhibits TarH-catalyzed ATP hydrolysis even though the binding site maps to TarG near the opposite side of the membrane. These are the first ABC transporter inhibitors shown to block ATPase activity by binding to the transmembrane domain. The compounds have potential as therapeutic agents to treat S. aureus infections, and purification of the transmembrane transporter will enable further development.

Full text of DOI:10.1021/jacs.7b04726

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Communication
An antibiotic that inhibits the ATPase activity of an ABC
transporter by binding to a remote extracellular site
Leigh Morgan Matano, Heidi Gabrielle Morris, Anthony R Hesser, Sara E. S. Martin, Wonsik Lee, Tristan W
Owens, Emaline Laney, Hidemasa Nakaminami, David Hooper, Timothy C. Meredith, and Suzanne Walker
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b04726 • Publication Date (Web): 20 Jul 2017
Downloaded from http://pubs.acs.org on July 20, 2017
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An antibiotic that inhibits the ATPase activity of an ABC transporter
by binding to a remote extracellular site
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Leigh M. Matano1◊, Heidi G. Morris1◊, Anthony R. Hesser1◊, Sara E. S. Martin1, Wonsik Lee1, Tristan
W. Owens2, Emaline Laney1, Hidemasa Nakaminami3, David Hooper3, Timothy C. Meredith4* and
Suzanne Walker1*
1Department of Microbiology and Immunobiology, 77 Ave Louis Pasteur, Boston, MA 02115, 2Department of Chemisꢀ
try and Chemical Biology, 12 Oxford St., Cambridge, MA 02138, 3Division of Infectious Diseases, Massachusetts
General Hospital, 55 Fruit St., Boston, MA 02114, 4Department of Biochemistry and Molecular Biology, 206 South
Frear Laboratory, University Park, PA 16802. ◊These authors contributed equally to the work.
Supporting Information Placeholder
porters are found in all domains of life and use ATP
binding and hydrolysis to power conformational changes
to translocate molecules across the cell membrane.4 Altꢀ
hough WTAs are required for infection,3a the first and
second steps in the biosynthetic pathway, catalyzed by
TarO and TarA, respectively, can be blocked genetically
or pharmacologically without loss of viability; however,
inhibiting subsequent steps is lethal and inhibitors of
these late steps have potential as antibiotics.5 We deꢀ
scribe here the discovery of a promising small molecule
that inhibits the wall teichoic acid pathway ABC transꢀ
porter and we show that it blocks the ATPase activity of
the nucleotide binding domain (NBD). Resistance mutaꢀ
tions map the binding site to the transmembrane domain.
Therefore, we propose that conformational
ABSTRACT: Antibioticꢀresistant strains of Staphylococcus
aureus pose a major threat to human health and there is
an ongoing need for new antibiotics to treat resistant
infections. In a high throughput screen (HTS) of 230,000
small molecules designed to identify bioactive wall
teichoic acid (WTA) inhibitors, we identified one hit,
which was expanded through chemical synthesis into a
small panel of potent compounds. We showed that these
compounds target TarG, the transmembrane component
of the twoꢀcomponent ATPꢀbinding cassette (ABC)
transporter TarGH, which exports WTA precursors to the
cell surface for attachment to peptidoglycan. We puriꢀ
fied, for the first time, a WTA transporter and have reꢀ
constituted ATPase activity in proteoliposomes. We
showed that this new compound series inhibits TarHꢀ
catalyzed ATP hydrolysis even though the binding site
maps to TarG near the opposite side of the membrane.
These are the first ABC transporter inhibitors shown to
block ATPase activity by binding to the transmembrane
domain. The compounds have potential as therapeutic
agents to treat S. aureus infections, and purification of
the transmembrane transporter will enable further develꢀ
opment.
Wall teichoic acid
Peptidoglycan
Vancomycin
Moenomycin
Lipoteichoic+
+
+
acid
Amsacrine
Bacitracin
Targocil
TarGH
+
+
D-ala
DltB
TarO
TarA
S. aureus has proven to be a highly adaptable
pathogen, developing resistance almost as quickly as
new antibiotics come to market.1 Maintaining a pipeline
of antibiotics with activity against S. aureus is necessary
to stay ahead of emerging resistance.2 The WTA pathꢀ
way is a promising antibacterial target because WTAs,
which are covalently attached to peptidoglycan, play
crucial roles in cell division, antibiotic resistance, and
pathogenesis.3 WTA precursors are synthesized on a liꢀ
pid carrier on the inner leaflet of the plasma membrane
and then exported to the cell surface by the two compoꢀ
nent ABC transporter TarGH (Figure 1).3b ABC transꢀ
Lipid II-Gly5 Lipid I
UDP-MurNAc-PP
Phospho-glycerol
Phospho-ribitol
UndP
UDP-GlcNAc
UDP-ManNAc
UndPP
Figure 1. Schematic of cell wall biosynthetic pathways
showing the sites of action of inhibitors mentioned in the
text. Blue arrows denote the peptidoglycan pathway and
red arrows denote the WTA pathway; these pathways use
the same undecaprenyl (UndP) carrier. Antibiotic structures
and legend abbreviations are explained in Figure S1.
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coupling between ABC transporter subunits can be exꢀ especially potent inhibitors of wildtype S. aureus growth
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ploited to develop specific inhibitors that can block acꢀ
tivity of the ATPase from a distance.
(0.125 µg/mL), but showed no activity against the ꢀtarO
strain (Figure 2B and Table S1). This MIC is eightꢀfold
lower than that of targocil, a wellꢀcharacterized WTAꢀ
active antibiotic.7a Moreover, the kinetic solubility of
these compounds is two to three logs greater than tarꢀ
gocil’s, the halfꢀlives were found to be 20ꢀ40 times
longer in mouse liver microsomes, and the compounds
were not cytotoxic (Table S2, Figure S2). Based on the
promising properties of the compound, we elucidated its
mechanism of action.
The lethal phenotype resulting from a late block
in the WTA pathway, which is due to depletion of peptiꢀ
doglycan precursors (see Figure 1),2c,6 inspired us to
develop a pathwayꢀspecific, whole cell assay for WTAꢀ
targeted antibiotics that involved screening a wildtype
strain for growth inhibition while counterscreening a
WTA null (ꢀtarO) strain for suppression of bioactivity.3b
As previously discovered WTA inhibitors had poor
physical properties,7 we screened 230,000 small moleꢀ
cules at a final concentration of ~15 µM against
wildtype S. aureus and the ꢀtarO knockout strain. The
screen produced a
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We first assessed the effect of the compound on
pool levels of the peptidoglycan precursor, Lipid II, usꢀ
ing a previously developed assay.2c,6 Compounds that
inhibit a late step in the wall teichoic acid pathway deꢀ
plete Lipid II because this peptidoglycan precursor is
biosynthesized on the same carrier lipid, undecaprenyl
phosphate (UndP, Figure 1).2c,5ꢀ6 If the UndP carrier lipid
is sequestered in WTA precursors, it is not available for
peptidoglycan precursor synthesis. Cultures of S. aureus
were treated for ten minutes with targocil, 2, or three
peptidoglycan synthesis inhibitors with mechanisms of
action that lead either to Lipid II depletion (bacitracin,
which inhibits carrier lipid recycling) or Lipid II accuꢀ
mulation (moenomycin and vancomycin, which inhibit
peptidoglycan assembly; Figure 1, Figure 3A). Cellular
lipids were extracted and the Lipid II present therein was
labeled with biotin to enable detection by streptavidinꢀ
HRP.2c,6 Like targocil and bacitracin, compound 2 deꢀ
pleted Lipid II. Combined with the suppression of bioacꢀ
tivity in the ꢀtarO strain, this result confirmed inhibition
of a late step in the WTA pathway.
To identify the molecular target within the
WTA pathway, we selected resistant mutants on comꢀ
pound 2. Twentyꢀseven colonies from three independent
cultures were selected for evaluation. We expected two
classes of mutants: those with mutations in the molecuꢀ
lar target and those with mutations that disrupted funcꢀ
tion of TarO or TarA.3b,5 To sort these mutants, we made
Figure 2. A HTS screening hit led to potent antiꢀMRSA
compounds 2 and 4. (A) Plot of HTS results. Each circle
represents the average OD600 of the strains in the presence
of a library compound tested in duplicate. One compound
(compound 1, red circle) inhibited growth of the WT
Newman strain but not ∆tarO. (B) Synthesized analogs of 1
with activities against S. aureus Newman. MICs against
MRSA strains are identical (Table S1).
use of the teichoic acid
Dꢀalanylation inhibitor, amsaꢀ
crine, which prevents growth of WTA null strains.9 Sevꢀ
enteen mutants were unable to grow on the inhibitor
(Figure 3B), and all of these were found to contain null
mutations in tarO or tarA; all other mutants contained
point mutations that resulted in amino acid substitutions
in TarG, the transmembrane component of the ABC
transporter that exports WTA precursors to the cell surꢀ
face (Figure 3C; Table S3). These results validated the
procedure used to classify mutants and suggested that
TarG is the target of 2.
single strong hit (1), which proved to be a furanocoumaꢀ
rin derivative (Figure 2A). Compound 1 was found to
have a minimum inhibitory concentration (MIC) of 1
µg/mL against S. aureus (Figure 2), including several βꢀ
lactam resistant strains (MRSA; Table S1). A literature
search revealed that compound 1 had been identified as
a growth inhibitor in a 2,000,000ꢀcompound screen for
S. aureus antibiotics, but its target was not identified.8
Based on structurally related compounds also reported in
that large screen, we synthesized a panel of analogs.
We used two different approaches to confirm
TarG as the target. First, we expressed one of the reꢀ
sistant tarG alleles in a clean S. aureus background and
found that expression conferred dominant resistance
(Figure S3). Second, after verifying that 2 did not inhibit
Two Lꢀproline derivatives (2 and 4) were found to be
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ported.11 Pꢀglycoprotein inhibitors have received the
growth of B. subtilis (Figure S4), we made use of a preꢀ
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viously engineered B. subtilis strain in which the endogꢀ
enous WTA transporter genes (tagGH) were replaced
with the S. aureus transporter genes at an ectopic
locus.10 Compound 2 did not show a zone of inhibition
in a disk diffusion assay against wildtype B. subtilis, but
it showed a doseꢀdependent inhibition zone when tested
against the strain expressing the S. aureus transporter
(Figure 3D). This gain of sensitivity to compound 2 upꢀ
on heterologous expression of S. aureus tarGH in B.
subtilis confirmed the S. aureus wall teichoic acid transꢀ
porter as its target.
most attention due to the importance of this ABC transꢀ
porter in multidrug resistance in cancer.12 Inhibitors that
compete with ATP for binding to the nucleotide binding
domain (NBD) or with exported substrates have been
studied, but were abandoned due to lack of specificity
and toxicity.12b The most promising Pꢀglycoprotein inꢀ
hibitors identified to date bind to the transmembrane
(TM) domain in a manner that prevents substrate
transport, but allows robust ATP hydrolysis.11c,13
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To obtain information on how 2 inhibits TarGH,
we coꢀexpressed wildtype TarG with either TarHꢀHis6 or
an ATPaseꢀinactive TarHꢀHis6 mutant (E169Q), solubilꢀ
ized the complexes in dodecylmaltoside, purified them
over an affinity column followed by size exclusion
chromatography, and reconstituted them into proteolipoꢀ
somes.14 The ATPase activity of the reconstituted transꢀ
porter, measured using a continuous chromogenic assay,
had kinetic parameters similar to those reported for other
ABC transporters (Figure S5).15 The addition of comꢀ
pound 2 strongly inhibited ATPase activity with an IC50
of 137 nM, even though the ATP concentration was
1000ꢀfold higher (Figure 4A, Figure S6). Additional
experiments showed that the ATPase activity of the WT
transporter in the presence of 1.0 µM 2 was comparable
to that of a TarGH mutant containing a mutation that
impairs ATP hydrolysis (TarH E169Q) (Figure S7; Figꢀ
ure S8).
To locate the binding site of 2 relative to the
ATPase, we generated a homology model for TarGH
using the human ABCG5/ABCG8 sterol transporter as
the template and mapped the resistance mutations to the
modeled structure (Figure 4B,C).16 In agreement with
the topology of many other ABC exporters, each TarGH
dimer has 12 TM helices,15b,17 which are grouped such
that TM helices one and two from one monomer are in
close proximity to TM helix five of the other monomer.
The highꢀlevel resistance mutations selected with comꢀ
pound 2 map near the extracellular ends of TM helices
one and five. While we cannot exclude the possibility
that the resistance mutations affect the conformation of
the ATPase from a distance such that it remains active
but is incapable of binding inhibitor, we think it far more
likely that the binding site is defined by the resistance
mutations. We propose, therefore, that the binding site
spans the dimer interface and, given the symmetry, that
two molecules of 2 bind to the dimer. To inhibit ATP
hydrolysis by binding to a remote site, the compound
must lock the TM domain in a conformation that preꢀ
vents the coupled interꢀdomain structural changes reꢀ
quired for ongoing ATP hydrolysis by the NBDs.
Figure 3. TarG is the target of 2. (A) Assay to detect Lipid
II abundance after antibiotic treatment, with results for conꢀ
trol antibiotics and 2 shown. Extracted Lipid II is labeled
with biotinꢀDꢀLys using S. aureus PBP4 to enable detection
with HRPꢀstreptavidin. (B) Mutants resistant to 2 (lanes 1ꢀ
3) were sorted into two groups by plating on amsacrine.
Susceptible mutants 1 and 2 had mutations in tarA while
amsacrineꢀresistant mutant 3 had a mutation in tarG (see
Table S3, S4 for full list and comparison to other TarG
inhibitors). (C) Substitutions in TarG that conferred high
level resistance to 2. (D) Disk diffusion assay shows that
strain KS002, in which B. subtilis TagGH was replaced
with S. aureus TarGH, is sensitive to 2.
Several classes of compounds that inhibit WTA
export have now been identified, but the class reported
here is the first with solubility properties that allow
mechanistic characterization.3b,7 Elucidating how these
compounds act may not only provide insight into how to
improve them further, but could guide efforts to develop
inhibitors of other ABC transporters. The ABC transꢀ
porter family is very large and includes many possible
therapeutic targets in both prokaryotes and eukaryotes,
but few mechanistic studies on inhibitors have been reꢀ
Compound 2, hereafter to be called targocilꢀII,
is the first known example of an ABC transporter inhibiꢀ
tor that prevents ATP hydrolysis by binding to an alloꢀ
steric site in the TM domain. Given the sequence diverꢀ
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sity of TM domains, this mode of binding would have
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clear advantages with respect to specificity over ATPꢀ
competitive inhibitors that bind to a very highly conꢀ
served binding pocket. Now that conditions have been
developed to obtain the purified ABC transporter in acꢀ
tive form, it may be possible to obtain structural inforꢀ
mation with inhibitor bound to facilitate development of
transport inhibitors for therapeutic use.
ACKNOWLEDGEMENTS
We thank: the staff at ICCBꢀLongwood Screening Facility
for help; the CETR DTR Core for toxicity testing; and Miꢀ
chael Cameron, Drug Metabolism and Pharmacokinetics
Core, Scripps Research Institute, for the microsomal stabilꢀ
ity and solubility assays.
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L195
TM2
F55
TarH
V54
TM1
Figure 4. Compound 2 inhibits the ATPase activity of
TarGH in proteoliposomes but binds in a remote location.
(A) Averaged ATPase activity (n=3; error bars=SD) of
reconstituted TarGH (200 nM) in the absence (black) and
presence (red) of compound 2 (1 ꢁM). Saturating levels of
ATP (1 mM) were used. (B) Homology model of TarGH.
TarH is cytoplasmic and much of TarG is embedded in the
membrane. C) Top view of the TarG dimer. Mutations in
residues shown in pink give high level resistance to 2.
ASSOCIATED CONTENT
Supporting Information. Supporting Information is availꢀ
able free of charge on the ACS Publications website. Methꢀ
ods, SI figures and tables (PDF).
AUTHOR INFORMATION
Corresponding Author
Suzanne Walker *suzanne_walker@hms.harvard.edu;
Timothy Meredith *txm50@psu.edu
Author Contributions
LMM, HGM and ARH contributed equally.
Funding Sources
No competing financial interests are declared. This work
was funded by NIH grants U19AI109764 and P01AI08324,
and NSF GRFP grant DGE1144152 (ARH).
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ACS Paragon Plus Environment