Comparison of small molecule inhibitors of the bacterial cell division protein ftsz and identification of a reliable cross-species inhibitor

Article abstract of DOI:10.1021/cb300340j

FtsZ is a guanosine triphosphatase (GTPase) that mediates cytokinesis in bacteria. FtsZ is homologous in structure to eukaryotic tubulin and polymerizes in a similar head-to-tail fashion. The study of tubulin's function in eukaryotic cells has benefited g

Full text of DOI:10.1021/cb300340j

Articles
pubs.acs.org/acschemicalbiology
Comparison of Small Molecule Inhibitors of the Bacterial Cell
Division Protein FtsZ and Identification of a Reliable Cross-Species
Inhibitor
David E. Anderson, Michelle B. Kim, Jared T. Moore, Terrence E. O’Brien, Nohemy A. Sorto,
Charles I. Grove, Laura L. Lackner, James B. Ames, and Jared T. Shaw*
Department of Chemistry, University of California, One Shields Ave, Davis, California 95616, United States
S
* Supporting Information
ABSTRACT: FtsZ is a guanosine triphosphatase (GTPase)
that mediates cytokinesis in bacteria. FtsZ is homologous in
structure to eukaryotic tubulin and polymerizes in a similar
head-to-tail fashion. The study of tubulin’s function in
eukaryotic cells has benefited greatly from specific and potent
small molecule inhibitors, including colchicine and taxol.
Although many small molecule inhibitors of FtsZ have been
reported, none has emerged as a generally useful probe for
modulating bacterial cell division. With the goal of establishing a
useful and reliable small molecule inhibitor of FtsZ, a broad biochemical cross-comparison of reported FtsZ inhibitors was
undertaken. Several of these molecules, including phenolic natural products, are unselective inhibitors that seem to derive their
activity from the formation of microscopic colloids or aggregates. Other compounds, including the natural product viriditoxin and
the drug development candidate PC190723, exhibit no inhibition of GTPase activity using protocols in this work or under
published conditions. Of the compounds studied, only zantrin Z3 exhibits good levels of inhibition, maintains activity under
conditions that disrupt small molecule aggregates, and provides a platform for exploration of structure−activity relationships
(SAR). Preliminary SAR studies have identified slight modifications to the two side chains of this structure that modulate the
inhibitory activity of zantrin Z3. Collectively, these studies will help focus future investigations toward the establishment of
probes for FtsZ that fill the roles of colchicine and taxol in studies of tubulin.
antibiotics has led to many reports of small molecule inhibitors.
ell division in bacteria is controlled by several proteins
Cthat make up the divisome in which FtsZ, the bacterial Given the close structural homology between FtsZ and tubulin,
the prospects for finding selective small molecule inhibitors has
always seemed good. Among the many potent inhibitors of
tubulin’s function, colchicine, taxol, and vinblastine all exhibit
selective effects that can be correlated to their molecular
interactions with the protein. In addition, the crystallographic
structures of tubulin bound to all three of these inhibitors5−7
enables new inhibitors to be classified by their similarity to
colchicine, which destabilizes tubulin polymers, and taxol,
which stabilizes polymers of tubulin. Both molecules are used as
drugs and as chemical probes for cell biology experiments.
Analogous information for FtsZ in bacteria is still lacking. None
of the known inhibitors exhibits potency that approaches that
of taxol’s and colchicine’s inhibition of tubulin’s function. Aside
from GTP and close analogues for which the structures of
complexes with FtsZ have been solved with X-ray crystallog-
raphy,8 there is little direct structural information for the basis
of perturbing FtsZ’s function with small molecules. The lone
example lies in the recent cocrystal structure of PC190723 with
homologue of eukaryotic tubulin, plays a central role (Figure
1).1−4 The possibility of modulating the activity of this protein
in order to better understand the cell division process in
prokaryotes and possibly advance FtsZ as a target of new
Received: July 9, 2012
Accepted: September 8, 2012
Published: September 8, 2012
Figure 1. FtsZ in bacterial cell division and possible pathways for
inhibition.
© 2012 American Chemical Society
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Figure 2. Small molecule inhibitors of FtsZ divided by structural class. (A) Cationic dyes, (B) nucleoside and nucleotide analogues, (C) drug-like
heterocycles, (D) phenolic natural products, (E) miscellaneous high-throughput screening hits, (F) anionic dye, (G) quaternary alkaloid natural
products.
FtsZ from Staphylococcus aureus (SaFtsZ), which confirms the
structural inferences made by resistant mutants of Bacillus
subtilis in the original disclosure of this inhibitor.9 The fact that
this compound preferentially affects SaFtsZ limits the extent to
which this result impacts the majority of studies that employ
FtsZ from Escherichia coli (EcFtsZ) and B. subtilis (BsFtsZ).
Importantly, there is little information for how well a small
molecule inhibitor of FtsZ from one species of bacteria inhibits
that from other species. This gap prevents both the
development of small molecule inhibitors of FtsZ as drugs
and the use of small molecules to better understand bacterial
cell division. Here, we report a critical assessment of small
molecule inhibitors of FtsZ to date and document that only a
small number currently represent good starting points for
finding small molecules that will be as useful to the study of
FtsZ as colchicine, taxol, and others have proven to the study of
tubulin.
The lack of generally useful FtsZ inhibitors might seem
strange, given that the literature would suggest that
investigators are surrounded by useful small molecule probes
of bacterial cell division that vary widely in their chemical
structures (Figure 2).10,11 Careful scrutiny of these molecules
suggests that they can be divided into seven groups based on
structure and origin. For instance, 4′,6-diamidino-2-phenyl-
indole (3, DAPI), a widely used DNA dye, resembles zantrins
Z5 (1) and Z2 (2) in that they all have extended heterocyclic
structures that are cationic at biological pH (Figure 2A).12,13
Compounds 48,14 and 515 were designed as GTP analogues
(Figure 2B). Although several different inhibitors (6−
10)13,16−23 resemble drug-like heterocyclic compounds, they
bear little structural resemblance to each other. It is notable that
PC190723 (8) and (1a-G7, 10) represent optimizations of
previously reported bacterial cell division inhibitors 3-
methoxybenzamide (3MBA)24 and thiabendazole, respectively
(not shown).25 Many FtsZ inhibitors have been isolated from
natural sources. The majority identified to date are nonalkaloid
structures that are almost all phenolic (Figure 2D).26−32
Similarly, screening of libraries of small molecules has provided
a wealth of structures united only by the ease of their chemical
synthesis or commercial availability (19−24, Figure
2E).13,19,33−36 Finally, the anionic dye bis-ANS (25) and two
quaternary alkaloids (26, 27) do not structurally resemble most
of the other compounds and are relegated to their own classes
(Figure 2F,G).37−39 The consistent traits of all of the molecules
in Figure 2 are that most have not been the subject of
additional studies since their discovery and that almost none
have been compared directly in biochemical or cell-based
assays.
Central to the problem of studying FtsZ with small molecule
inhibitors is cross-species reactivity, i.e., the extent to which an
inhibitor reported for protein from one species of bacteria
might affect the activity of protein from another species. Most
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inhibitors reported to date have documented activity for
protein from one species of bacterium, usually E. coli or B.
subtilis. A small number of experiments are reported for S.
aureus and M. tuberculosis FtsZ, the latter of which is known to
be a much slower enzyme and, as a result, generally requires
higher concentrations of inhibitor and protein. Confounding
the issue further is the multitude of assays for the function of
FtsZ. The most consistent assay is for GTP hydrolysis, which is
usually measured either by an enzyme-coupled assay that
consumes NADH or by the detection of free phosphate with
malachite green. The latter assay has largely replaced the use of
radiolabeled GTP in studies of FtsZ. Displacement of GTP
analogues, including the fluorescent mant-GTP40 and GTP-γ-
S,41 has also been reported. Finally, the interpretation of
biochemical studies is complicated by differences in buffers and
additives that prevent comparison between results from
different laboratories. Another problem is the complex coupling
between GTPase activity and filament formation. The direct
impact of small molecules on polymerization can be examined
with electron microscopy, light scattering, or centrifugation to
separate free FtsZ from polymers, or so-called protofilaments. A
highly selective inhibitor would be expected to phenocopy the
temperature sensitive mutants of FtsZ from which many of the
genes controlling cell division derive their name (fts,
filamentous temperature sensitive). With that said, assessing
the chemically induced phenotype is complicated by the many
other mechanisms by which filamentation might be induced,
including (1) induction of the SOS response by genotoxic
compounds, (2) inhibition of one or more of the other cell
division proteins and (3) decoupling of the Z-ring from the cell
wall, which can be induced by several different classes of small
molecules.42 Furthermore, the inability of bacteria to function
without FtsZ prohibits the examination of mutants that lack
this protein, which is a standard control experiment in chemical
biology by which one can assign the target of a small molecule.
Collectively, these caveats greatly limit the extent to which
induction of filamentation can be used as evidence for selective
chemical inhibition of FtsZ or any other cell division protein.
We set out to discover new and better inhibitors of FtsZ with
the long-term goal of ascertaining the molecular interactions by
which these compounds affect FtsZ using NMR or X-ray
crystallography. As a starting point, we needed to be able to
compare and contrast any new compounds with known
inhibitors that we assumed would help establish structure−
activity relationships (SAR) and enable the rational design of
compounds to synthesize. Among the compounds in Figure 2,
we opted not to study compounds with known liabilities
associated with low activity or promiscuity and instead focused
on a subset of nine representative compounds with the
potential for further refinement. Close examination of the
activity of these compounds has revealed some important
conclusions that will impact future studies of small molecule
inhibitors of FtsZ. First, there is compelling evidence that many
inhibitors derive their activity from the formation of aggregates,
as described by Shoichet.43 Second, several reported inhibitors
do not exhibit the reported levels of inhibition when examined
under standardized conditions. Third, zantrin Z3 (9) was found
to be a robust and reliable inhibitor of FtsZ from two different
species (E. coli and B. subtilis) and it does not appear to form
aggregates that result in unselective inhibition. Synthetic
analogues of 9 exhibit informative SAR, with one in particular
exhibiting enhanced biochemical inhibition of FtsZ. These data
cement this compound as an effective reagent to use as a
positive control and as a starting point for our search for better
inhibitors. Herein, we describe all of the experiments that led to
these conclusions and, hopefully, establish consistent protocols
for assaying future inhibitors so that useful comparisons across
FtsZ from various organisms are straightforward.
RESULTS AND DISCUSSION
■
Identification of FtsZ Inhibitors That Likely Form
Aggregates. Small molecules discovered in high-throughput
screens sometimes exhibit enzyme inhibitory activity that
results from the formation of aggregates or colloids.44,45
Shoichet has documented many cases of this effect46−48 and
established experiments for assessing the presence of
aggregates.43,49 We have been interested in the activity of
totarol, which has a long history of antimicrobial activity50,51
and was recently shown to inhibit FtsZ.28 Using the enzyme-
coupled GTPase assay, we were able to reproduce the IC50 of
totarol (Figure 3). Oddly, several close analogues of totarol
Figure 3. Inhibition of GTPase activity of FtsZ by totarol (17), zantrin
Z1 (24), and 4′-hydroxydichamanetin (15b) in the absence and
presence of 0.01% Triton X-100: (A) BsFtsZ and (B) EcFtsZ.
showed absolutely no inhibition of FtsZ in the same assay (data
not shown). We suspected that this dichotomy could be due to
the formation of aggregates and repeated the assay in the
presence of 0.01% Triton X-100, a neutral detergent shown to
disrupt aggregate formation in enzymatic assays. In the
presence of Triton X-100, totarol’s activity vanished. Similar
behavior was observed when a structurally unrelated eukaryotic
GTPase was examined. Dnm1, which mediates mitochondrial
division,52,53 showed significant reduction in GTPase activity in
the presence of micromolar concentrations of totarol, and the
activity was completely restored by the addition of 0.01%
Triton X-100 (see Supporting Information). Similar reduction
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in FtsZ’s activity was observed for 4′-hydroxydichamanetin
(4HD) and zantrin Z1, each of which exhibited the published
activity in the absence of Triton X-100. In our control
experiments, i.e., those in which no inhibitor is added, GTPase
activity remained constant in the presence or absence of Triton
X-100, indicating that the protein was largely unaffected by this
low concentration of detergent. Dichamanetin was substituted
with 4HD, which is prepared in a single synthetic step from
commercially available materials and behaves identically to
dichamanetin (See Supporting Information).
In order to confirm aggregate formation as the underlying
mechanism for inhibition of FtsZ by totarol, several additional
experiments were conducted. Stock solutions of totarol in
buffer were centrifuged before adding aliquots to the assay wells
(Figure 4). Again, no activity was observed, consistent with the
Figure 5. Inhibition of FtsZ GTPase activity by 24 μM and 64 μM
totarol at increasing FtsZ concentrations: (A) BsFtsZ and (B) EcFtsZ.
The slight downward trend for DMSO only can be explained by
increased bundling of FtsZ protofilaments at the higher concen-
trations, which can lead to slower nucleotide exchange.73
Figure 4. Inhibition of EcFtsZ and BsFtsZ GTPase activity by totarol
before and after centrifugation of the drug in assay buffer.
ability of small molecule colloids to be removed by
centrifugation. Next, we varied the concentration of protein
and noticed that, as the concentration increased, the inhibitory
activity of totarol decreased nearly to the point of having no
effect well above the established IC50 (Figure 5). This result is
consistent with the formation of particles that are becoming
saturated with protein as opposed to soluble small molecules
that are still present in gross molar excess relative to the
protein. Collectively, these results support the conclusion that
totarol’s inhibition of FtsZ is an artifact of aggregate formation.
Although the additional experiments were not performed on
dichamanetin and zantrin Z1, the Triton experiment provides
strong evidence in favor of aggregate formation, and we
conclude that all future reports of FtsZ inhibition by small
molecules should include this important control experiment.
On the basis of the structural appearance and similar bulk
properties, zantrin Z4 (22) and sulfoalkylresorcinol 14 should
be viewed as aggregation suspects until proven otherwise.
Screening compound PC170942, which is structurally
unrelated to PC190723 (see Figure 2), exhibits unusual
behavior consistent with some level of aggregation. Under
standard conditions, an IC50 of approximately 160 μM is
observed, which is 3-fold higher than originally reported
(Figure 6). In the presence of 0.01% Triton X-100, inhibition is
similar, and oddly, the IC50 is slightly lower. The enzyme-
coupled assay was used for this experiment, and it was noted
that inhibition seemed to consistently decrease over time when
examining the level of enzymatic activity at longer time points.
We hypothesized that this effect was due to slow deaggregation
of PC170942 by Triton X-100. Aliquots of the compound were
treated with the detergent and stored for 45 min before
Figure 6. Inhibition of BsFtsZ activity by PC170942 in the absence of
Triton X-100, with 0.01% Triton X-100, and after 45 min incubation
with 0.01% Triton X-100.
repeating the dose response. The IC50 was shifted significantly
higher, suggesting that the primary mode of inhibition was by
the formation of small molecule aggregates. This result suggests
that, in some cases, longer incubation times for the Triton X-
100 control experiment might be necessary to completely rule
out unselective inhibition by aggregates.
Assessment of Benzamides (8J and PC190723),
Viriditoxin, SRI-3072, and Zantrin Z3. The search for new
small molecule inhibitors relies on the ability to make
comparisons to positive controls, i.e., compounds that are
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known to inhibit FtsZ to a certain degree under an established
set of conditions. We initially looked to PC190723, given that
this is the compound with the lowest reported IC50 and
allegedly the best known inhibitor of FtsZ GTPase activity. We
reported a practical synthesis of this compound in order to
ensure a steady supply for our laboratory and others with whom
we have shared this reagent.54 Although the reported activity is
for S. aureus FtsZ, the close analogue 8J (7) was shown to also
inhibit BsFtsZ.55 We observed no inhibition of B. subtilis FtsZ
by PC190723 at concentrations up to 100 μM using either the
enzyme-coupled or malachite green assays (Figure 7). The
PC190723, this compound was examined in B. subtilis, and
inhibition of FtsZ from that organism was reported,55 albeit
without documenting the specific activity of the enzyme. We
observed no inhibitory activity using the enzyme-coupled assay
with up to 128 μM of 8J (Figure 8). Direct comparison was
Figure 8. Effect of 8J (7) on the GTPase activity of BsFtsZ using the
enzyme-coupled and malachite green assays.
Figure 7. Effect of PC190723 on SaFtsZ and BsFtsZ as observed by
enzyme-coupled and malachite green assays for GTPase activity. Basal
FtsZ GTPase activity (no inhibitor) was 0.5 FtsZ/GTP/min for
SaFtsZ and 4.4 FtsZ/GTP/min for BsFtsZ.
possible by switching to the malachite green assay. During the
optimization of this assay for this protein, a variety of different
responses were observed. On one occasion, there was a dose-
dependent reduction in activity, albeit with similarly large error
bars when compared to the published data, shallow sloping of
the dose response curve, and only 50% reduction in enzymatic
activity (data not shown). In our case, this was apparently due
to incomplete quenching of the reactions and unequal times on
ice before malachite green reagent addition. The most
consistently observed behavior for 8J was a slight dose-
dependent increase in enzymatic activity. This trend was
observed reproducibly with either the malachite green or
enzyme-coupled assays, with slight variation between the two
for the magnitude of the increase. Compound 8J exhibited
better antimicrobial activity than PC190723 against B. subtilis
strain 168 (MIC 0.25 μM), consistent with the reported value
for S. aureus.22
Viriditoxin is one of the most potent inhibitors from a natural
source reported to date with an IC50 of 10 μM. Our lab had
previously completed a synthesis of this compound and
confirmed the identity by isolating this metabolite from a
culture of Aspergillus viridinutans.58 Under standard conditions,
no inhibition of FtsZ was observed up to a concentration of 200
μM for either B. subtilis or E. coli FtsZ. Careful inspection of the
report from Merck revealed unique buffer conditions that
included only 40 μM GTP, 0.75 mM Mg(OAc)2, and 1 μM
FtsZ at a pH of 7.4. In addition, the authors included 2.5 mM
CaCl2 and 50 μg/mL DEAE-Dextran, which favor extensive
FtsZ polymerization in order to facilitate rapid separation of
polymeric FtsZ from monomers and short oligomers in a filter
binding assay.59 On the basis of the premise that the activity of
viriditoxin could only be observed under these unusual
conditions, we attempted to replicate the assay that is
documented in the original report and still observed no
inhibition at concentrations up to 128 μM. In this second
experiment, the baseline enzymatic activity (i.e., in the absence
of inhibitor) was reduced by 2 orders of magnitude (from 5
FtsZ/GTP/min down to 0.05, see Supporting Information),
latter result conflicts with the observations of Andreu and co-
workers who observed widely variant activity, including dose-
dependent increase or decrease of enzymatic activity, while
studying the biochemical effects of PC190723 on BsFtsZ and
EcFtsZ.56 As a point of direct comparison to the original work
of Haydon et al., the activity of PC190723 was examined for S.
aureus FtsZ (SaFtsZ), and still no inhibition was observed. In
fact, a dose-dependent and reproducible increase in activity for
SaFtsZ was observed (Figure 7). In this case, the basal activity
of the enzyme is significantly lower (∼0.5 GTP/FtsZ/min)
than we observed for EcFtsZ or BsFtsZ. The original report
includes only the relative change in activity (as a percentage of
control) without noting the actual enzymatic turnover in the
absence of inhibitor. The activity of BsFtsZ and EcFtsZ can also
vary greatly depending on conditions (vide infra), making these
data important when assessing inhibition of the enzymatic
function. The lack of reproducibility of PC190723’s inhibition
of SaFtsZ has been confirmed in at least one other laboratory to
whom we have provided this compound (data not shown).
Very recently (i.e., during the review of this article), a detailed
study of the mechanism of action of PC190723 by scientists at
Merck documents this compound as an activator of the GTPase
activity of SaFtsZ, consistent with our results.57 We did observe
antimicrobial activity against B. subtilis strain 168 that closely
matched the original report (MIC 1 μM).20 Although we
currently have no explanation for these results, it can be safely
concluded that PC190723 cannot serve as a positive control for
in vitro experiments examining the GTPase activity of FtsZ
from S. aureus or any other organism.
Compound 8J was also examined as a point of direct
comparison. This compound differs from PC190723 by a single
atom substitution (carbon for nitrogen on the heterocyclic
appendage) involving replacement of a pyridine ring in the side
chain with a benzene ring. As a followup to the studies of
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which greatly decreases the signal-to-noise of any biochemical
experiment under these conditions. We cannot rule out the
possibility that viriditoxin may inhibit the GTPase activity of
FtsZ or otherwise interfere with the function of this protein
under a very specific and not completely documented set of
conditions. Furthermore, we have noted that viriditoxin is
chemically unstable and degrades if it is not stored free of
solvent and at low temperature. The toxicity of this compound
prevents further development of this compound for drug
discovery, and our data indicates that it is also not useful as a
biochemical probe for SAR studies.
SRI-3072 was reported in a study that focused specifically on
inhibitors of M. tuberculosis FtsZ (MtbFtsZ). The GTPase
activity of this protein is significantly lower than that of E. coli
or B. subitilis, which is probably related, on some level, to the
slow division of this organism. Recent SAR studies on this
compound identified a few analogues that have lower IC50
values for FtsZ inhibition, but in all cases, the MICs were
higher for two strains of M. tuberculosis, and in one case, a
modest level of tubulin inhibition was observed. The effects of
these compounds on FtsZ were mostly studied by light
scattering, and in one case, the GTPase activity was shown to
decrease by 20% at 100 μM. Given the 5−10-fold higher
concentrations at which the zantrins inhibit MtbFtsZ relative to
EcFtsZ, it was possible that significant inhibition of the latter
protein might be possible at a much lower concentration. This
compound exhibited no inhibition of E. coli or B. subtilis FtsZ
up to 128 μM (see Supporting Information).
Zantrin Z3, along with the other zantrins, was discovered in a
high-throughput screen for inhibitors of the GTPase activity of
FtsZ. In spite of the shared name for these compounds, their
chemical structures are largely unrelated except in the cases of
zantrins Z3 and Z5, which are both structurally similar to DAPI
(Figure 2). Zantrin Z3 stands out for having a structure that is
drug-like, i.e., it has no offending electrophilic or phenolic
functionality and has a passing resemblance to the antimalarial
drug chloroquine. Inhibition of EcFtsZ was observed, and the
IC50 value (20 μM) compares favorably with the reported value
(Figure 9). Zantrin Z3 also effectively inhibited the function of
Figure 10. Inhibition of FtsZ GTPase activity by 15 μM and 50 μM
zantrin Z3 at increasing FtsZ concentrations: (A) BsFtsZ and (B)
EcFtsZ.
We have developed a modular synthesis of zantrin Z3 and
conducted preliminary structure−activity relationship (SAR)
studies (Figure 11). Although an exact synthesis for zantrin Z3
has not been published, the route employed was adapted from
Okano and co-workers.60 Naphthopyrimidinone 28 is made in
a single step from commercially available 3-amino-2-naphthoic
Figure 9. Inhibition of GTPase activity of BsFtsZ and EcFtsZ by
zantrin Z3 in the absence and presence of 0.01% Triton X-100.
BsFtsZ with an IC50 of 32 μM. Inhibition was maintained in the
presence of 0.01% Triton X-100 (Figure 9) and at increasing
concentrations of protein (Figure 10). Collectively, these
experiments suggest that the activity is probably not an artifact
of aggregate formation. Of all the compounds examined in this
study, zantrin Z3 is the best in terms of overall performance
against protein from multiple species of bacteria under a variety
of conditions.
Figure 11. Synthesis of zantrin Z3 analogues and IC50s for inhibition
of EcFtsZ.
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acid. This compound can be carried through an aldol-like
condensation with a variety of benzaldehydes to provide
compounds 29a−e in modest to good yields. These
intermediates were converted to their corresponding chlorides
and then treated with various N,N-dialkylaminoethylamines.
Variation of the styryl side chain revealed that the para-
substituent significantly influenced the activity and that
halogens (F, Cl, and Br) were all superior to H or methyl.
Replacement of the aminoethyl side chain with a less basic (35)
or nonbasic (34) side chain abolished the inhibition observed
with the other side chains. Finally, replacement of the
diethylamino group with a dimethylamino group resulted in
the best inhibitor of the series (38, 12 μM). This compound is
currently being used for cocrystallization with BsFtsZ and
EcFtsZ as well as isolation of resistant strains of E. coli and B.
subtilis. Current efforts are directed at replacing the styryl side
chain with a different linker that will enhance the inhibitory
activity.
Implications for Future Studies of FtsZ Inhibition.
Zantrin Z3 and the related compounds with similar activity (31
and 37) are uniquely active and selective in their inhibition of
FtsZ when compared to the other molecules examined in this
study or described previously. Many of the compounds in
Figure 2 were not examined based on established liabilities.
One of the first inhibitors was bis-ANS, a sulfonate dye
molecule37 (25, Figure 2), known to occupy nucleotide binding
sites on several proteins, including myosin61 and tubulin.62
DAPI (3), a dye used for staining double-stranded DNA, has
been reported as an inhibitor of FtsZ,12 and the structural
similarity to two other zantrins (Z2/5 and Z5/1)13 suggested
these two could be similarly promiscuous. Two nucleotide
analogues have been documented as inhibitors of FtsZ. The
first, 8-methoxy GTP (4), inhibits the GTPase activity of FtsZ
at 15 μM and a crystal structure of a related compound in the
series (8-morpholino-GTP, not shown) documents binding of
this compound in the active site of FtsZ.8 Although all of the
GTP analogues discussed in this study showed some level of
inhibition of FtsZ GTP hydrolysis, the compounds were
roughly split between analogues that promote or inhibit tubulin
assembly. Finally, although they were not examined in this
study, the extremely high IC50 values for GAL10 (5, 450 μM)15
and A189 (21, 260 μM)33 test the limits of solubility in
aqueous media.
Some FtsZ inhibitors are not specific and modulate the
function of other proteins. OTBA (23)34 and a recently
disclosed analogue (not shown) share a common core
(rhodanine) that is included in a list of pan-assay interference
compounds (PAINS) due to the promiscuity with which
certain recurrent compounds appear as hits in high-throughput
screening experiments.63 The fact that OTBA has an IC50 for
FtsZ of approximately 20 μM and is cytotoxic to mammalian
cells at 8 μM is consistent with PAIN behavior. Although
zantrin Z3 may have additional targets, the substructure of this
molecule is not among the PAINS.
Natural product inhibitors of FtsZ all carry significant
liabilities to further development as selective probes, which is
surprising given the natural origin of the best tubulin inhibitors
(e.g., taxol, colchicine, and vinblastine). Curcumin (18)32 and
cinnamaldehyde (13)29 have both been described as FtsZ
inhibitors but also as inhibitors of many other proteins and
pathways, including tubulin in the case of curcumin.64
Dichamanetin (15)65 and HBB (16)66 are antimicrobial natural
products whose close structural similarity to zantrin Z1 (24)
suggested that they also inhibited FtsZ, and their activities were
reported to be 12.5 and 8.3 μM, respectively.31 Our studies
demonstrate that these compounds, along with totarol, are all
probably inhibiting FtsZ by forming aggregates and call into
question the origin of the inhibitory activity of sulfoalkylre-
sorcinol derivative 14 and the recently isolated chrysophaentin
A.27 Although viriditoxin (11) was found in a high-throughput
screen for compounds that disrupted polymerization of FtsZ
tagged with a fluorescent reporter,26 this activity is not easily
replicated using more standard conditions. Two cationic
alkaloid natural products, berberine (27)38 and sanguinarine
(26),39 also inhibit the function of FtsZ. The latter compound
is also known to inhibit tubulin assembly, whereas the former
does not do so, even at high concentration (100 μM).67,68
Finally, a series of taxol derivatives (not shown) was designed
and synthesized on the premise that the similar structures
between FtsZ and tubulin would result in similar structures of
inhibitors.69 These compounds are described as FtsZ inhibitors
solely on the filamented phenotype that is observed by bacteria
treated with the compounds. Given the large number of
mechanisms that can result in filamentation, conclusions
regarding a single protein target based solely on this (or any)
phenotype should be made cautiously. All told, Mother Nature
has been far less generous with selective and potent inhibitors
of FtsZ when compared to tubulin.
The activity of PC190723, the only submicromolar inhibitor
of FtsZ GTPase activity reported to date, is the most difficult to
interpret in light of the subsequent studies of this molecule and
its close analogue 8J. The original publication does not include
a detailed protocol for the assay. Although one was eventually
provided by one of the authors (N. Stokes), the specific activity
of the SaFtsZ was not recorded, thus making it difficult to
directly compare our results with theirs. Subsequent reports
from Andreu56 and Errington55 on PC190723 and 8J,
respectively, using EcFtsZ and/or BsFtsZ do not contain any
obvious explanation for the discrepancy in reported and
observed inhibition of SaFtsZ. In our studies, the activity of
SaFtsZ is quite low, making any interpretation of its
biochemical behavior more error-prone than analogous studies
with EcFtsZ or BsFtsZ. However, an interaction between
PC190723 with SaFtsZ is clearly indicated by the recent
publication of the cocrystal structure,55 which is the only
structure of FtsZ published to date in a complex with a non-
nucleotide small molecule. Moreover, this structure confirms
the binding region predicted by the original resistant mutant
studies. We have indeed observed antimicrobial activity of
PC190723 against B. subtilis that was very similar to that
originally reported. The conclusion from all of these data is that
the behavior of FtsZ in vitro can be highly erratic and assay-
dependent, even in cases where all other indicators point to
strong interaction that disrupts cell division.
Zantrin Z3′s biochemical inhibition of FtsZ makes it an
important probe for future investigations of the bacterial cell
division machinery. Demonstration that this molecule does not
inhibit FtsZ through an unselective aggregation mechanism is
an important first step in understanding how inhibition of the
GTPase activity of FtsZ impacts bacterial cell division. Ideally, a
compound with significantly better inhibition will be identified
in future SAR studies for both cell-based studies and
crystallography. The activity against EcFtsZ, BsFtsZ, and
MtFtsZ13 makes this a useful compound for studying all of
these organisms, on which the vast majority of studies to date
are based. Although FtsZ still lacks the potent and selective
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thoroughly mixed, and 150 μL of each reaction was pipetted into a
microplate well. Absorbance readings were taken over time at room
temperature (RT) (∼25 °C) on a Biotek EL808 microplate reader at
340 nm.
small molecule tools available for studying tubulin, these studies
demonstrate a clear path forward for discovery of new FtsZ
inhibitors.
Malachite Green GTPase Assay. This assay was performed
approximately as described13 without the addition of sodium citrate
due to the high amounts of phosphates known to contaminate many
sources of citrate. FtsZ from E. coli or B. subtilis (2 μM) or S. aureus
(10 μM) was mixed with a 50× concentrate of the inhibitor (4 μL) in
50 mM MES buffer, pH 6.5, 5 mM Mg(OAc)2, and 100 mM KOAc to
a final volume of 198 μL and incubated for 5 min. GTP hydrolysis was
initiated with 1 mM GTP (2 μL) to give a final volume of 200 μL. At
appropriate time points after incubation at 30 °C, 20 μL of the
reaction mixture was transferred to a tube containing 5 μL of 100 mM
EDTA on ice. Twenty microliters of a series of phosphate standards
were also transferred into EDTA on ice. Then, 600 μL of freshly
prepared malachite green working reagent [2 volumes malachite green
(0.8 mg mL−1)/1 volume polyvinyl alcohol (23.2 mg mL−1)/1 volume
ammonium molybdate (57.2 mg mL−1 in 2H2O:3HCl)/2 volumes
H2O, allowed to sit for 30 min] was added to each tube, mixed, and
incubated for 30 min at RT. Finally, 200 μL of each sample was
pipetted into a microplate well and absorbance readings were taken on
a Biotek EL808 microplate reader at 630 nm.
Approximate Recapitulation of Merck Viriditoxin Condi-
tions. To repeat as closely as possible the reported GTPase inhibition
by viriditoxin,26 without using a radioactive assay as reported, we used
the malachite green assay described above under the following reaction
conditions: 50 mM Tris, 34 mM KCl, 0.75 mM Mg(OAc)2, 2.5 mM
CaCl2, and 50 μg mL−1 DEAE-Dextran (Sigma D9885) at 37 °C.
Curve Fitting of IC50 Data. Inhibition data was fit to a logistic
equation, y = A2 + (A1 − A2)/(1 + (x/x0)p), by the method of least-
squares, using the Solver function of Microsoft Excel and allowing all
parameters (A1, A2, x0, p) to vary. Occasionally, the lower asymptote/
maximally inhibited value was fixed at 0 if the first solution gave a
negative number. Several iterations were performed to confirm
convergence.
METHODS
■
Protein Expression and Purification. E. coli FtsZ in pET11b was
the kind gift of Harold Erickson (Duke University). B. subtilis FtsZ was
cloned from B. subtilis strain 168 by colony PCR using primers 5′-
AATTAACATATGTTGGAGTTCGAAACAAACATAG-3′ and 5′-
ATAGGATCCTTAGCCGCGTTTATTACGGTTTC-3′. The FtsZ
PCR fragment was purified, digested with NdeI and BamHI, then
ligated into NdeI/BamHI-digested pET11b plasmid. The final product
was verified by sequencing. S. aureus FtsZ in pET11a was the kind gift
of Carole Bewley (NIH/NIDDK).
C41(DE3) cells (Lucigen) were used as the host strain for protein
expression. Cells were grown at 37 °C in LB with 100 μg mL−1
ampicillin to an OD (600 nm) of approximately 0.6, then induced with
0.5 mM IPTG for 3 h at 37 °C, with the exception of S. aureus FtsZ,
which was induced at 30 °C for 5 h. Cells were pelleted at 6000 rpm in
a FIBERLite F9-4x1000y rotor (Thermo Scientific) and either frozen
overnight before resuspension or directly resuspended in 50 mM Tris,
1 mM EDTA, 50 mM KCl, 10% (v/v) glycerol, pH 7.9 (Buffer A).
Resuspended cells were treated with lysozyme (Sigma-Aldrich) for 30
min−1 h on ice, then lysed by probe sonication for 1.5 min on ice (5
min with 30% duty cycle). The lysate was clarified by centrifugation at
35 000 rpm in a Sorvall T-1250 rotor for 1 h at 4 °C, and the resulting
supernatant brought to 35%-saturated (Ec, Bs) or 50%-saturated (Sa)
ammonium sulfate. After incubation at 0 °C for at least 30 min with
periodic mixing, precipitated protein was collected by centrifugation at
20 000 rpm in a Sorvall SS-34 rotor for 20 min at 4 °C. Ammonium
sulfate pellets were resuspended in Buffer A lacking KCl and dialyzed
against the same buffer before loading onto a HiPrep Q Sepharose
FastFlow anion exchange column (GE Healthcare Life Sciences).
Fractions were eluted with a 0−800 mM KCl gradient and analyzed by
SDS-PAGE. Peak FtsZ fractions were aliquotted and stored at −80 °C.
Prior to enzymatic assays, FtsZ protein was dialyzed into the
appropriate assay buffer and the concentration determined using the
BCA protein assay (Thermo Fisher). The calculated concentration of
FtsZ was divided by 0.75 (∼1.33-fold increase in estimated
concentration) due to the lower color development of FtsZ relative
to BSA in the assay.70 Specific activity ranged from 4−6 GTP/FtsZ/
min for BsFtsZ and from 3−5 GTP/FtsZ/min for EcFtsZ. The specific
activity for SaFtsZ was more variable and generally lower, ranging from
0.1 to 1 GTP/FtsZ/min.
Small Molecule Inhibitors. Small molecule inhibitors were either
purchased or synthesized as described in the Supporting Information.
Zantrin Z1 (24) was purchased from Ryan Scientific. Although zantrin
Z3 (9) was initially purchased from Ryan Scientific, it was later
discontinued and was synthesized by the route developed for
compounds 30−38 (see Supporting Information). 4HD (15b) was
prepared in analogy to dichamanetin31 using naringenin as a starting
material. Compound 8j (7) was prepared in analogy to PC190723 (see
Supporting Information). SRI-3072 was kindly provided by Dr. Robert
Reynolds (Southern Research Institute). The synthesis of PC170942
(19) was not reported in the literature, and the route developed for
this work is described in the Supporting Information. Viriditoxin (12)
and PC190723 (8) were prepared by previously described
routes.54,58,71
Aggregation Tests. When testing for compound aggregation43 in
the GTPase assay, the following conditions were used: for detergent
addition, 0.01% (v/v) Triton X-100 (from a freshly prepared 1% (v/v)
stock) was added at the beginning along with buffer and compound
stock; for centrifugation, the buffer/compound mixture was centri-
fuged at 14 100g for 20 min before addition of protein; for increasing
enzyme concentration, the amount of FtsZ was varied as noted in the
corresponding figure.
ASSOCIATED CONTENT
* Supporting Information
■
S
Additional details for biochemical experiments; syntheses of 8J,
PC190723, PC170942, zantrin Z3 and compounds 30−38.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: jtshaw@ucdavis.edu.
■
Notes
The authors declare no competing financial interest.
Enzyme-Coupled GTPase Assay. This assay was calibrated and
performed generally as described.72 Specifically, a 50× concentrate of
the inhibitor in DMSO (4 μL) was mixed into the appropriate volume
of assay buffer (50 mM MES buffer, pH 6.5, 5 mM Mg(OAc)2, and
100 mM KOAc), then FtsZ from E. coli or B. subtilis (2 μM) or S.
aureus (10 μM) was added and incubated at 37 °C for 5 min. Next, 40
μL of a 5× “master mix” of coupling enzymes and substrates
(containing 100 U mL−1 each of lactate dehydrogenase/pyruvate
kinase, 2.5 mM phosphoenolpyruvate, and 2.0 mM NADH, all from
Sigma-Aldrich) was added. GTP hydrolysis was initiated with 1 mM
GTP (2 μL) to give a final volume of 200 μL. The solution was
ACKNOWLEDGMENTS
■
This work was supported by the National Institutes of Heath/
National Institute of Allergy and Infectious Diseases (NIH/
NIAID) (R01AI08093), National Institutes of Health/ Na-
tional Eye Institute (EY012347) to JBA, and by National
Institutes of Heath/National Institute of General Medical
Sciences (NIH/NIGMS) (T32-GM008799), which provided
J.T.M. with predoctoral fellowship support. M.B.K. and T.E.O.
acknowledge support in the form of GAANN fellowships.
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T.E.O. and J.T.M. acknowledge support from Bradford Borge
fellowships from UC Davis. N.A.S. acknowledges fellowship
support from Bristol-Meyers Squibb and from Proctor and
Gamble through the National GEM consortium. N.A.S., T.E.O.,
and C.I.G. were also supported by fellowships from the Alfred
P. Sloan Foundation. C. Bewley (National Institutes of Heath/
National Institute of Diabetes and Digestive and Kidney
Diseases, NIH/NIDDK) is acknowledged for helpful dis-
cussions, conducting confirmatory experiments with
PC190723, and for providing the plasmid for SaFtsZ. H.
Erickson (Duke University) is acknowledged for providing the
plasmid for EcFtsZ. R. Reynolds (Southern Research Institute)
is acknowledged for providing a sample of SRI-3072 (6).
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