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Transcript
Perspective
pubs.acs.org/jmc
Targeting the Bacterial Division Protein FtsZ
Katherine A. Hurley,†,# Thiago M. A. Santos,‡,# Gabriella M. Nepomuceno,§ Valerie Huynh,§
Jared T. Shaw,*,§ and Douglas B. Weibel*,‡,∥,⊥
†
Department of Pharmaceutical Sciences, University of WisconsinMadison, 777 Highland Avenue, Madison, Wisconsin 53705,
United States
‡
Department of Biochemistry, University of WisconsinMadison, 440 Henry Mall, Madison, Wisconsin 53706, United States
§
Department of Chemistry, University of CaliforniaDavis, One Shields Avenue, Davis, California 95616, United States
∥
Department of Chemistry, University of WisconsinMadison, 1101 University Avenue, Madison, Wisconsin 53706, United States
⊥
Department of Biomedical Engineering, University of WisconsinMadison, 1550 Engineering Drive, Madison, Wisconsin 53706,
United States
ABSTRACT: Similar to its eukaryotic counterpart, the prokaryotic cytoskeleton is essential for the structural and mechanical
properties of bacterial cells. The essential protein FtsZ is a central player in the cytoskeletal family, forms a cytokinetic ring
at mid-cell, and recruits the division machinery to orchestrate cell division. Cells depleted of or lacking functional FtsZ do
not divide and grow into long filaments that eventually lyse. FtsZ has been studied extensively as a target for antibacterial
development. In this Perspective, we review the structural and biochemical properties of FtsZ, its role in cell biochemistry and
physiology, the different mechanisms of inhibiting FtsZ, small molecule antagonists (including some misconceptions about
mechanisms of action), and their discovery strategies. This collective information will inform chemists on different aspects of
FtsZ that can be (and have been) used to develop successful strategies for devising new families of cell division inhibitors.
1. INTRODUCTION: TARGETING THE BACTERIAL
PROTEIN FtsZ
An increase of multidrug resistance to antibiotics among
pathogenic strains of bacteria and the lack of innovation in
the discovery of new antibacterial agents punctuate the need
for new chemotherapeutic strategies. One approach to new
strategies is the identification, characterization, and exploration
of new molecular targets for antibiotic development, which is
currently in vogue. Historically, all known clinical antibiotics
target one of the following bacterial structures and cellular
processes: (1) DNA replication; (2) transcription; (3) translation; (4) peptidoglycan biosynthesis; (5) folate biosynthesis;
(6) the cytoplasmic membrane.1,2 An important, unanswered
question is whether additional classes of mechanisms and
targets exist for developing new families of antibiotics. The
bacterial cytoskeleton is one such family of targets for which
clinical antibiotics have not yet emerged.
The cytoskeleton is an ancient cellular invention that probably
precedes the divergence between eukaryotes and prokaryotes.3
The bacterial cytoskeleton consists of families of proteins essential
for the physiological and structural properties of cells, including
cell division,4,5 cell wall growth,6,7 cell shape determination/
maintenance,8,9 DNA segregation,10 and protein localization10
(Table 1). Because its integrity is important to cell viability, the
bacterial cytoskeleton has been a topic of discussion for the
development of antibacterial compounds over the past 2 decades.
© 2016 American Chemical Society
The essential cytoskeletal cell division protein FtsZ (named
after the filamenting temperature-sensitive mutant Z) is an
essential GTPase structurally related to eukaryotic tubulins11−13
and highly conserved in bacteria and archaea.14,15 During cell
division, FtsZ forms a ringlike structure at the site of division and
functions as a scaffold for the assembly of a multiprotein complex
(referred to as the “divisome”) essential for cell viability.
Not surprisingly, FtsZ, as well as proteins that interact
directly with and regulate the activity of FtsZ, has emerged as a
prime target for antibacterial development.16 The use of FtsZ as
an antibacterial drug target has been reviewed,17,18 and its
structural biology16,19,20 and inhibition with small molecules
have been discussed.21−25 Specifically, targeting FtsZ with small
molecules as a defense against tuberculosis has also been
extensively reviewed.26−28 In this review, we explore the latest
developments of classes of small molecules and inhibitors
targeting FtsZ and evaluate the challenges and future directions
of this field of antibiotic research.
2. STRUCTURE AND FUNCTION OF FtsZ
2.1. FtsZ Structure. FtsZ shares 40−50% sequence identity
across most bacterial and archaeal species and has a threedimensional structure that is similar to the structure of α- and
Received: July 14, 2015
Published: January 12, 2016
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DOI: 10.1021/acs.jmedchem.5b01098
J. Med. Chem. 2016, 59, 6975−6998
Journal of Medicinal Chemistry
Perspective
Table 1. Examples of Key Components of the Bacterial Cytoskeletona
cytoskeletal proteinb
Tubulin-like
FtsZ
TubZ
Actin-like
FtsA
MreB
ParM
Intermediate Filaments
crescentin
Walker A “Cytoskeletal” ATPases
MinD
ParA
function/remarks
-Is the structural subunit of the Z-ring
-Recruits downstream proteins involved in cell division and cell wall synthesis and remodeling
-Involved in DNA segregation
-Membrane tether required for Z-ring assembly
-Recruits downstream proteins involved in cell division and cell wall synthesis and remodeling
-Destabilizes FtsZ filaments on the membrane, enabling rapid reorganization of the filament network222
-Required for cell shape determination (morphogenesis) and maintenance
-Is also implicated in chromosome segregation and cell polarity
-Participates in DNA segregation
-Responsible for the asymmetric cell shape in some bacteria (e.g., it is an essential determinant of the curved shapes of C. crescentus cells)
-Involved in positioning the Z-ring at mid-cell
-Participates in DNA segregation
a
This is not a comprehensive list. The bacterial cytoskeleton consists of other families of proteins or protein homologues that are absent from this
list. Further information can be found reviewed in ref 223. bSome of these cytoskeletal proteins are essential and widespread among bacteria.
However, some of them are exclusive to specific bacteria groups. See the text and refer to the cited literature for an additional explanation.
The referencing is not exhaustive for the best-studied proteins included in the list.
Figure 1. FtsZ is the ancestral homologue of tubulin and is highly conserved in bacteria. Top: A representation of the monomers of FtsZ and
β-tubulin with GDP (in orange) bound in the active site. Left to right: S. aureus (PBD code 3VOA),85 M. jannaschii (PBD code 1FSZ),29 and
S. scrofa (PBD code 1TUB).30 The direction of the polymerization is indicated based on the axis of the protofilament. Bottom left: A representation
of the dimerization of two monomers of FtsZ from S. aureus (PBD code 3VOA)85 and M. jannaschii (PBD code 1W5A)29 with GDP (in red) bound
in the active site. Each monomer is represented as a different shade of green to facilitate visualization, and GDP is represented as electrostatic spheres
in brick red. Bottom right: A demonstration of the dimerization of one monomer of β-tubulin (dark green) with GDP bound in the active site and
one monomer of α-tubulin (light green) with GTP bound in the active site from S. scrofa (PBD code 1TUB).30 The direction of the polymerization
is indicated based on the axis of the protofilament. These representations were generated using PyMOL (version 1.5.0.4).
β-tubulin3,13,29,30 (Figure 1). Despite structural and functional
similarities, FtsZ is a distant ancestral homolog of tubulin with
an amino acid sequence that is <20% identical.3,13,31,32
Crystallographic analysis of FtsZ from the hyperthermophilic
methanogen Methanocaldococcus jannaschii (formerly Methanococcus jannaschii) revealed the presence of two domains
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Figure 2. Spatiotemporal regulation of the Z-ring in different groups of bacteria. (A) Diagram summarizing the hierarchical recruitment of cell
division proteins in E. coli. FtsZ and the early cell division proteins localize to the division site before cell septation starts. The proteins are recruited
to the Z-ring in a sequential and approximately linear pathway. The requirement of an upstream protein for localization of a downstream protein to
the Z-ring was deduced from various studies of genetics, biochemistry, and microscopy. Proteins that regulate the assembly of the Z-ring are shown
in green (positive regulators) and red (negative regulators). Peptidoglycan-specific amidases AmiA, AmiB, and AmiC play an important role in
cleaving the septum to release daughter cells after division in E. coli. AmiB and AmiC localize to the division site, whereas AmiA (not included in the
diagram) is diffusely localized in the periplasm.216 This diagram was redrawn from refs 48, 51, and 217. (B) A cartoon illustrating some of the
bacterial mechanisms for positioning of the Z-ring during cell division. In E. coli, Min proteins (MinCDE) oscillate between the cell poles, creating an
inhibition zone (green shaded area) and preventing Z-ring (in red) polymerization near those poles.60,61,218 (For simplicity, the dynamic behavior of
the Min system is omitted.) In addition, nucleoid occlusion, mediated by the protein SlmA, creates an inhibition zone (blue shaded area) along the
cylindrical region of the cell and prevents Z-ring assembly over the nucleoid.63 Inset: A cartoon depicting the predicted organization of the E. coli
divisome. Cell division is initiated with the polymerization of FtsZ into the Z-ring onto which the divisome apparatus assembles. The cartoon of the
divisome was adapted from refs 48 and 217. Similar to E. coli, the B. subtilis Min system (composed of MinCDJ and DivIVA) creates a zone of
inhibition (purple shaded area) that prevents Z-ring assembly at the cell poles. However, in B. subtilis, MinCDJ localizes to the cell poles in a DivIVAdependent manner and does not undergo the characteristic dynamic oscillatory behavior observed in E. coli.219,220 In addition to the Min system, the
protein Noc mediates nucleoid occlusion (blue shaded area), preventing divisome assembly from occurring over segregating chromosomes.62
In C. crescentus, the protein MipZ (yellow shaded area) coordinates chromosome segregation and cell division in response to both spatial and
temporal cues. The assembly of Z-ring is coincident with the subcellular position that exhibits the lowest concentration of MipZ. Prior to
chromosome replication, MipZ and FtsZ are localized to the opposite poles of the cell. MipZ forms a complex with proteins involved in chromosome
partitioning. Following duplication of the chromosomal replication origin region (oriC), MipZ migrates toward the opposite cell pole creating a
bipolar gradient displacing the FtsZ at the poles and directing formation of the Z-ring toward mid-cell.65,221 In S. pneumoniae, the protein MapZ
forms a ringlike structure (in orange) positioned at mid-cell (and at future division sites), marking the cell division site and positioning FtsZ.66
See the text for an additional explanation.
connected by a long central helix (H7)13 (also designated as helix
H529) (Figure 1). The amino-terminal portion of the protein
consists of a six-stranded β-sheet sandwiched by two helices on
one side and three on the other and contains the GTPase domain.
This domain is conserved between FtsZ and tubulin; however, it is
different in other classic GTPases.13 The carboxy-terminal domain
consists of a four-stranded β-sheet in contact with helix H7 and
supported by two helices on one side while the other side is
exposed to the solvent.29 The conserved C-terminal tail of FtsZ
mediates specific interactions between FtsZ and auxiliary proteins
that regulate divisome assembly and disassembly, such as MinC,33
FtsA,34,35 ZipA,34−36 EzrA,37 ClpX,38 SepF,39 and FtsZ itself.40
FtsZ assembles into protofilaments that form tubules, sheets,
and minirings in vitro.41,42 Super-resolution microscopy studies
in vivo demonstrate that the Escherichia coli Z-ring adopts a
compressed helical conformation with variable helical length
and thickness of ∼110 nm.43 Electron cryotomographic reconstructions of dividing Caulobacter crescentus cells revealed that
the Z-ring consists of short protofilaments that are ∼100 nm
in length and randomly spaced near the division site and
positioned ∼16 nm away from the inner membrane.44 More
recent electron cryomicroscopic and cryotomographic studies
in E. coli, C. crescentus, and constricting liposomes confirmed
the distance of the protofilaments positioned from the inner
membrane and revealed that in both bacterial species the Z-ring
is probably a continuous structure consisted of single-layered
bundles of FtsZ that are 5−10 filaments wide.45
2.2. Role of FtsZ in Cell Division. Bacterial cell division
is a complex process that requires accurate identification of
the division site, positioning of the division machinery, and
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B. subtilis. However, many bacteria lack both canonical systems
for positioning FtsZ. For example, in the Gram-negative
bacterium C. crescentus, spatiotemporal assembly and placement
of the Z-ring require MipZ.65 MipZ is an ATPase that associates with the origin region of chromosomes and (in a manner
analogous to the Min system) directly guides FtsZ positioning
and polymerization into the Z-ring at mid-cell65 (Figure 2B).
In the Gram-positive pathogen Streptococcus pneumoniae, MapZ
localizes at the division site prior to FtsZ, interacts directly with
FtsZ, and guides positioning of the Z-ring66 (Figure 2B). MapZ
is a single-passage transmembrane protein that is conserved
among Streptococcaceae and other Lactobacillales and forms
ringlike structures positioned at mid-cell and at future division
sites. In addition to its dual role in marking the cell division site
and positioning FtsZ, the balance between the phosphorylated
and dephosphorylated forms of MapZ may be important for
controlling Z-ring stability and regulation of constriction.66
In addition to these specific protein-based systems that
control FtsZ dynamics, other positive and negative regulators
interact with FtsZ to modulate structure and function of the
divisome in response to the nutritional and developmental state
of the cell (Table 2). These regulatory systems for positioning
of FtsZ have adapted to different environments, cell shapes, and
developmental behaviors and emphasize the importance of
coordinating the correct timing of cell division and chromosome
segregation. Many studies have indicated that unknown
regulators of bacterial FtsZ may be awaiting discovery.
2.5. FtsZ Biophysics and Mechanics. An average E. coli
cell during log-phase growth contains ∼15 000 molecules of
FtsZ.67 The Z-rings assemble at the future site of division in
E. coli daughter cells before the Z-ring is fully constricted in the
parental cell.59 This observation suggests that future division
sites in daughter cells become competent for assembly of the
divisome prior to the complete division of the mother cell. In
addition, FRAP experiments in E. coli cells demonstrate that the
Z-ring is a dynamic structure, continuously remodeled by
exchanging subunits with the cytoplasmic pool of FtsZ (halftime of recovery ∼30 s). The kinetics of FtsZ turnover in vivo is
tightly coupled to GTP hydrolysis, as mutant cells with reduced
GTPase activity of FtsZ show ∼9-fold slower turnover of
FtsZ into protofilaments.68 In vitro studies performed at
physiological conditions show that purified E. coli FtsZ
assembles into protofilaments and hydrolyze GTP at a rate of
∼5 molecules per min per FtsZ, demonstrating that the
GTPase activity of FtsZ in vitro is very slow.67
In addition to functioning as an essential molecular scaffold
for recruitment and organization of the other cell division
proteins to the division site,48,69 FtsZ generates a contractile
force that constricts tubular liposomes in vitro.70−72 FtsZ is
thought to act as an important source of the constriction force
required for cytokinesis during cell division; however, the
mechanism by which FtsZ generates mechanical force and
promotes invagination of the cell wall during division remains
unclear. Previous studies with purified FtsZ have shown that
the GTP-bound FtsZ assembles into straight or gently curved
filaments, while the GDP-bound FtsZ forms highly curved
filaments,41,42 suggesting that the difference in the intrinsic
curvature of FtsZ filaments provides a mechanism for generating mechanical force for cell division.
Models describing the process of cell growth and Z-ring
contraction in E. coli cells predict that a force of ∼8 pN is
sufficient to pull the cell wall inward at the division site to
initiate the constriction, and forces of 8−80 pN could lead to cell
coordinated constriction of the inner membrane and the cell
wall (i.e., cytokinesis). With few exceptions,46,47 this essential
process is initiated with the polymerization of FtsZ into a
filamentous, ringlike structure (referred to as the Z-ring) that is
located in the cytoplasm peripheral to the membrane and close
to the division site.5,43−45 Concomitant with and following its
polymerization, the Z-ring recruits and coordinates a series
of auxiliary proteins that perform diverse roles in cell division
and cell wall biosynthesis and remodeling48−51 (Figure 2A).
Depletion of FtsZ in rod-shaped bacteria, such as the Gramnegative E. coli, produces long, filamentous cells due to the
continued growth of cells that are no longer dividing.4
Cocci-shaped bacteria, such as the Gram-positive pathogen
Staphylococcus aureus, increase in volume up to 8-fold when
depleted of FtsZ.52 In both cases, cells are unable to divide;
continued growth makes them enlarged and sensitive to
changes in the physical properties of their environment, and
the cells eventually lyse. Drugs that affect the positioning,
activity, and interaction of FtsZ with other division proteins
cause cell lysis and may be useful as antibiotics.
2.3. FtsZ Dynamics during the Division Cycle. The
spatiotemporal regulation of Z-ring formation requires a
complex and concerted network of proteins that modulate
assembly and activity of FtsZ to ensure that the division process
is tightly coordinated with DNA replication, chromosome
segregation, and cell elongation.48,53−56 The molecular details
underlying the synchronicity of these processes are not
completely understood; however, structural and cell biology
research over the past 2 decades has elucidated important structural, functional, and regulatory aspects of these mechanisms
and how they are coordinated.
The division process in E. coli cells requires at least 14 major
cytoplasmic, membrane, and periplasmic proteins, of which
∼10 are essential48,57,58 (Figure 2A). FtsZ and the other cell
division proteins, as well as their regulators, are recruited to
mid-cell in a hierarchical order to form the functional divisome,
a ringlike multiprotein complex that constricts during the process of division and disappears when the cells separate49,50,57,59
(Figure 2B). The divisome machinery is essential and appears
to be widely conserved among bacteria.
2.4. Regulatory Proteins That Position FtsZ in
Bacteria. Rod-shape bacteria (such as E. coli and the Grampositive bacterium Bacillus subtilis) use at least two coordinated
biochemical systems to accurately position the Z-ring at the
mid-cell and ensure that divisome formation is timed to occur
at the final stage of the cell cycle: (1) the Min system of
proteins prevents aberrant division at regions other than the
mid-cell60,61 and (2) nucleoid occlusion proteins prevent
division from occurring over segregating chromosomes62,63
(Figure 2B). Importantly, in the absence of these two negative
regulators of Z-ring positioning, both E. coli and B. subtilis still
have a bias for Z-ring formation at mid-cell,62,63 suggesting
that additional mechanisms may influence FtsZ assembly at the
mid-cell and coordinate chromosome segregation and cell
division. A recent study provided evidence of an additional
positional marker in E. coli cells grown in minimal media and
lacking functional Min and nucleoid occlusion systems.64 In
particular, the authors identified that the Ter macrodomain
region of the chromosome acts as a landmark for the Z-ring
in the presence of the chromosomal terminus organization
protein MatP and the cell division proteins ZapA and ZapB.64
Much of the mechanistic insight on the spatiotemporal
regulation of the Z-ring has come from studies of E. coli and
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Table 2. Proteins That Regulate the Formation of the Z-Ring in Bacteriaa
(1) Positive Regulators of Z-Ring Formation51,69
b
FtsA
SepF
ZapA,B,C,D
ZipA
ClpX/ClpXP
CrgA
EzrA
GdhZ
KidO
OpgH
MciZ
MinC
MipZ
MapZ
Noc
SlmA
SulA
UgtP
YneA
-Membrane anchor supports assembly and stabilization of the Z-ring. It is also important for the recruitment of downstream proteins necessary for divisome
maturation.
-Required for proper morphology of the divisional septum. It has overlapping roles with FtsA in Z-ring assembly.
-Mediates additional stabilization of the Z-ring.
-Secondary membrane anchor that together with FtsA supports assembly of the Z-ring.
(2) Negative Regulators of Z-Ring Formation
-Helps modulate the equilibrium between the cytoplasmic pool of unassembled FtsZ and polymeric FtsZ through degradation.38 ClpX chaperone can also inhibit
formation of the Z-ring in a ClpP-independent fashion by physically blocking the assembly of FtsZ filaments.153−155
-Important for coordinating cell growth and division. It regulates the dynamics of Z-ring formation and affects both the timing of FtsZ expression and its
turnover.224
-Modulates the position of the Z-ring during cell division and plays a role in coordinating cell growth and division.69
-NAD-dependent glutamate dehydrogenase that controls Z-ring disassembly by stimulating the GTPase activity of FtsZ.225
-Coordinates cellular or developmental activities with the availability of NADH. KidO bound to NADH is thought to destabilize lateral interactions between
FtsZ protofilaments. It has been recently proposed to work in synergy with GdhZ to trigger Z-ring disassembly.225,226
-Glucosyltransferase that functions as a nutrient-dependent antagonist of the Z-ring. OpgH is thought to sequester FtsZ from growing polymers. Blocks Z-ring
formation to coordinate cell growth and cell division.227
-Inhibits Z-ring formation by capping the minus end of FtsZ filaments and shortening the filaments.228
-Important for positioning the Z-ring at mid-cell.60,61,218
-Required for positioning the Z-ring at mid-cell.65
-Important for Z-ring formation and positioning at mid-cell. It is also involved in the regulation of cytokinesis.66
-Inhibits Z-ring formation over segregating chromosomes.62
-Analogous to Noc, it inhibits Z-ring formation over segregating chromosomes.63
-Negative modulator of Z-ring expressed in response to DNA damage as part of the SOS system.165,168,169
-Similar to OpgH, this glucosyltransferase inhibits cell division by blocking Z-ring formation in a growth rate-dependent fashion. This cellular sensor ensures that
cells reach the appropriate mass and complete chromosome segregation prior to cytokinesis.229
-Analogous to SulA, it regulates cell division through the suppression of Z-ring formation during the SOS response.230
a
Some of these proteins and the molecular systems that they compose are widespread among bacteria. However, some of them are exclusive to
specific groups of bacteria. See the text and refer to the cited literature for an additional explanation. The referencing is not exhaustive for the beststudied proteins listed. bIt was recently demonstrated that FtsA has a dual, antagonistic role on the FtsZ filament network. FtsA is involved in
recruitment of FtsZ filaments to the membrane, but it also provides a negative regulation by causing fragmentation of FtsZ polymers, allowing the
rapid disassembly of FtsZ filaments.222
division by creating a reasonably accurate septal morphology.73
Molecular simulations of FtsZ dynamics estimate that FtsZ can
generate a value of ∼20−30 pN per polymerized monomer
when GTP is hydrolyzed,74 which is sufficient to direct cell−
wall invagination during division and cause membrane vesicle
formation from liposomes in vitro.70−72,75 Although mathematical models quantitatively support the “hydrolyze-and-bend”
mechanism for force generation of the Z-ring,41,76,77 slightly
curved GTP-FtsZ filaments (without GTP-hydrolysis) are
capable of supplying a force of ∼10 pN, suggesting that
nucleotide hydrolysis might not be required for membrane
bending by FtsZ.74,75 This observation could explain the
occasional division events observed in cells containing a mutant
version of FtsZ that has very low GTPase activity78−80 and the
initial constriction of tubular liposomes in the presence of this
“GTPase-dead” mutant FtsZ.75
2.6. FtsZ and Cell Morphology. The peptidoglycan layer
of bacterial cell walls consists of a heteropolymer of
polysaccharides cross-linked with short peptides that function
as the load-bearing material to resist mechanical and physical
forces (e.g., osmotic pressure) on cells. During the growth of
rod-shaped cells, peptidoglycan is assembled in two distinct
regions of the cell: (1) along the cylindrical body of cells, which
is required for cell elongation, and (2) at the site of cell
division, which creates a new curved pole for the two daughter
cells.
FtsZ is required for septal/cell-division-associated peptidoglycan growth and remodeling due to its essential role in
recruiting cell-division-specific peptidoglycan synthesis enzymes.49,50 However, recent research suggests that the regulatory role of FtsZ on peptidoglycan synthesis during cell
division extends beyond its ability to recruit proteins to the
mid-cell.7 Particularly, it was recently shown that the intrinsically
disordered C-terminal linker region of FtsZ is important for
regulation of enzymes involved in peptidoglycan metabolism in
C. crescentus.7
In addition to septal/cell-division-associated peptidoglycan
growth mediated by the divisome, rod-shaped bacteria have
other cellular machinery mediating lateral peptidoglycan
synthesis along the length of the cell. This multiprotein
complex named the “elongasome” is organized by the ancestral
homologue of actin, MreB.49 Until recently, the role of FtsZ
has been thought to be restricted to participating in peptidoglycan assembly and remodeling at the division site.
However, recent studies have demonstrated that FtsZ may
also play a role in elongation-associated cell wall growth in rodshape bacteria.6,81,82
The direct role of an FtsZ homologue in cell shape control of
rod-shaped microorganisms has been also demonstrated in
archaea. Unlike most bacteria, archaeal genomes frequently
contain additional genes belonging to the FtsZ/tubulin
superfamily.83 The archaeal tubulin-like protein CetZ, formerly
annotated as “FtsZ3” or “FtsZ type 2”, has been implicated in
cell shape control of Haloferax volcanii.84 CetZ has the FtsZ/
tubulin superfamily fold and a crystal form containing sheets of
protofilaments that suggest it may play a structural role in cells.
Inactivation of CetZ1 in H. volcanii does not affect cell division;
however, it prevents differentiation of the irregular plate-shaped
cells into a rod-shaped cell type essential for normal swimming
motility. CetZ1 forms dynamic cytoskeletal structures in vivo,
indicating its capacity to remodel the cell envelope and direct
rod formation.84
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Journal of Medicinal Chemistry
Perspective
3. MECHANISMS OF ACTION OF INHIBITORS OF FtsZ
Several characteristics validate FtsZ as a target for the development of new antibiotics to selectively combat bacterial infections: (1) it is essential and plays a specific role in prokaryotic
cell division;4 (2) it is structurally and functionally conserved
across bacterial and archaeal species;15,29,85 (3) although
widespread in mitochondria of diverse protist lineages, it is
notably absent in higher eukaryotes;83,86 (4) it is evolutionarily
distant from its eukaryotic counterpart tubulin;3,13,31,32 and
(5) there is a growing body of research on its structural,
biochemical, and biological properties.
3.1. Antagonism of Polymerization and GTPase
Activity of FtsZ by Small Molecules. Hydrolysis of GTP
requires assembly of two FtsZ monomers to complete the
catalytic site. This innate step in catalysis can be modulated by
targeting either the T7 loop of the “upper” monomer or the
nucleotide-binding pocket of the “lower” monomer.87 Alternatively, an allosteric site on FtsZ may modulate its ability to
form protofilaments. In this sense, the tightly regulated division
process could be halted by several mechanisms, including
(1) overly stabilizing protofilaments, which cannot disassemble
as GDP is produced by GTP hydrolysis; (2) destabilizing protofilaments; and (3) preventing polymerization. The chemical
inhibitors of FtsZ reported to date can be classified into three
main groups: (1) natural products and their derivatives;
(2) nucleotide analogs; and (3) molecules that emerged from
high-throughput screening. Below, we provide an overview of
the inhibitors that were a starting point for further development
of structurally related compounds or assays for their activity as
inhibitors of cell division. However, a caveat to this list is that
many of these inhibitors have been shown to be false positives
or to have irreproducible activity. This section delineates the
reported, albeit limited, mechanistic detail of reported FtsZ
inhibitors, which lays the foundation for a discussion of validated inhibitors in section 4. A summary of the antimicrobial
activity, discovery methods, and FtsZ binding characteristics of
these compounds is presented in Table 3.
3.1.1. FtsZ Inhibitors from Natural Products: Alkaloids.
Sanguinarine (1) is a polycyclic alkaloid that inhibits FtsZ
protofilament assembly by decreasing FtsZ polymerization;88 it
also inhibits eukaryotic tubulin, which complicates its use as an
antibiotic. Berberine (2) is a structurally related alkaloid that
inhibits GTPase activity and decreases FtsZ polymerization.
It is predicted to bind in the vicinity of the GTP binding pocket
and overlaps with several hydrophobic residues located in the
GTP binding site.89 Although allegedly indifferent to tubulin, 2
has since been described as a promiscuous binder of different
proteins.90 Berberine 2 (3) was designed to have an extended
alkyl group in place of one of the methyl groups on 2; the in
vitro GTPase inhibition activity of 3 was measured to be
approximately 38 μM against S. aureus FtsZ.91
3.1.2. FtsZ Inhibitors from Natural Products: Polyphenols.
Plumbagin (4) inhibits the GTPase activity of FtsZ and increases the lag phase of FtsZ assembly (i.e., adversely affects the
nucleation rate). The predicted binding site of 4 is located close
to the C-terminal domain of FtsZ in a region of the H7 helix,
spatially distant from the GTP binding domain.92 SA-011 (5)93 was
synthesized as an analog of 4 and shown to inhibit the GTPase
activity of Bacillus anthracis slightly better than 2. Resveratrol (6)
has been screened many times due to its known antimicrobial
activity, which has been attributed to inhibiting Z-ring formation
and suppressing the expression of FtsZ mRNA.92,94
Dichamanetin (7) and 2‴-hydroxy-5″-benzylisouvarinol-B
(8) are structurally similar pinocembrin-based molecules that
inhibit the GTPase activity of FtsZ in Gram-positive bacteria. 8
also displays antimicrobial activity against E. coli and
Pseudomonas aeruginosa.95 However, 7 was later shown to be
an “aggregator”, a molecule that forms aggregates that bind
nonspecifically to proteins. Viriditoxin (9) was initially reported
to inhibit the GTPase activity of FtsZ and cause cells to
filament, while overexpression of FtsZ was shown to rescue
drug-treated cells.96 9 has since been confirmed as a falsepositive that has activity that has not been reproducible.97 The
complex natural product family of chrysophaentins (e.g.,
chrysophaentin A (10)) was shown to inhibit the GTPase
activity of E. coli and S. aureus FtsZ (including methicillinresistant S. aureus strains), and molecular docking experiments
showed that the compound occludes a large portion of the
GTP binding site of the protein.98 FtsZ polymerization is
inhibited, and the Z-ring is mislocalized in cells treated with 10.
Despite these results, cell filamentation was not observed in a
mutant strain of E. coli (envA1) permeable to a wide variety of
compounds.99
3.1.3. FtsZ Inhibitors from Natural Products: Phenylpropanoids and Terpenoids. Several phenylpropanoids that
are derived from cinnamaldehyde (11) or related structures
have been tested for antimicrobial activity. Nearly all phenylpropanoids described as FtsZ inhibitors to date are alleged to
interact with at least one residue of the T7 loop.100 Virtual
screens and/or docking experiments of many of these
structurally “simple” natural products suggest they have specific
interactions with FtsZ. However, few examples have translated
into reliable inhibitors and lack biophysical support for
targeting FtsZ.
11 inhibits the GTPase activity of FtsZ, decreases polymerization and is not toxic to red blood cells.101 Phenylacrylamide
14 (12)102 has antibacterial activity against S. aureus and
Streptococcus pyogenes and inhibited cell division in S. aureus.
Vanillin derivatives 3a (13)103 and 4u (14)104 have been
independently tested against Mycobacterium tuberculosis FtsZ.
Scopoletin (15), a coumarin analog related to esculetin and
quercetin, inhibits the GTPase activity and polymerization of
FtsZ into protofilaments.105 Curcumin (16) increases the
GTPase activity and destabilizes polymerization of FtsZ, thus
reducing the steady-state duration of polymer assembly.106
Unlike other phenylpropanoids, the predicted FtsZ binding site
of 16 involves residues connected to GTP binding of FtsZ.107
Colchicine (17), although highly active against tubulin polymerization, has also been tested against FtsZ and has been
demonstrated to have no effect on FtsZ polymerization.108
Sulfoalkylresorcinol (18) inhibits the GTPase activity of
FtsZ in vitro and exhibits antimicrobial activity against various
pathogens but is not cytotoxic toward human A549 cells.109
Synthetic derivative n-undecyl gallate (19) disrupts ZapA
localization and possibly Z-ring formation in Xanthomonas citri
subsp. citri.110
Totarol (20) is a terpenoid that inhibits the GTPase activity
and polymerization of FtsZ protofilaments. Cells treated with
20 become filamentous and display a mislocalized Z-ring.111 20
was initially described as lacking activity against eukaryotic
tubulin; more recently it has been shown to be promiscuous in
binding to proteins and to have properties consistent with
being an aggregator.97 Germacrene D (21) and germacrene
D-4-ol (22) are part of a family of terpenoids isolated from the
essential oil of pine needles that exhibit antibacterial activity on
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Table 3. Summary of Reported FtsZ Inhibitors Discussed in the Texta
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Table 3. continued
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Table 3. continued
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Table 3. continued
a
Ec = Escherichia coli; EcFtsZ = recombinant E. coli FtsZ; SaFtsZ = recombinant Staphylococcus aureus FtsZ; BsFtsZ = recombinant Bacillus subtilis
FtsZ; MtFtZ = recombinant Mycobacterium tuberculosis FtsZ; PaFtsZ = recombinant Pseudomonas aeruginosa FtsZ; BaFtsZ = recombinant Bacillus
anthracis FtsZ; MRSA = methicillin-resistant Staphylococcus aureus
core and altered oxidation pattern (denoted as TRA 10a (25)
or 10b (26)) improved the potency of this taxane-derived
structure to target M. tuberculosis FtsZ.114
3.1.5. FtsZ Inhibitors That Mimic Nucleosides. Nucleotide
analogs have been explored as competitive inhibitors of GTP
for binding to FtsZ. 8-Bromoguanosine 5′-triphosphate (27)
binds to FtsZ with a Ki of 32 μM and inhibits both FtsZ polymerization and GTPase activity.115 Gal cores 10 (28), 14 (29),
and 15 (30)116 were designed to mimic the sugar-phosphate
backbone of GTP and shown to inhibit the GTPase activity of
P. aeruginosa FtsZ through an enzyme-coupled assay.
3.1.6. FtsZ Inhibitors from High-Throughput Screening of
Chemical Libraries. A number of structurally distinct small
molecules emerged from in vivo high-throughput screens as
causing cell filamentation and were considered to be targeting
FtsZ. Quinoline 1 (31)117 was screened against M. tuberculosis
various species of bacteria. A docking model predicts a binding
site of the germacrene family to be a hydrophobic pocket in
FtsZ; however, the crystallographic evidence for this interaction
is not yet determined.112
3.1.4. FtsZ Inhibitors That Are Derived from Taxanes.
Taxane-derived structures have been successfully modified to
target FtsZ preferentially over its eukaryotic homologue
tubulin. The taxane polycyclic core in these compounds has
remained largely intact. For example, SB-RA-2001 (23)113 only
differs from paclitaxel (24) in two ways: (1) the alcohol at C-10
lacks an acetyl group, and (2) an unsaturated ester has replaced
the α-hydroxy-β-amido ester of 24 at C-13. These structural
changes led to inhibition of enzymatic activity against B. subtilis
FtsZ and were shown to have antimicrobial activity against
both B. subtilis and Mycobacterium smegmatis. Huang et al. later
showed that the conjugated ester coupled with a ring-opened
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Compound 297F (49)135 is the most structurally divergent
compound in the benzamide family of FtsZ inhibitors and
targets M. tuberculosis; however, it bears little structural
resemblance to 42.
3.1.8. FtsZ Inhibitors Based on a Benzimidazole Scaffold.
A series of novel trisubstituted benzimidazoles, which were
inspired by tubulin-targeting thiabendazole (50) and albendazole (51),136 have been reported as targeting M. tuberculosis
FtsZ (Mtb-FtsZ). 137−142 Two benzaimidazoles, 1a-G7
(SB-P3G2) (52) and 1a-G4 (53),137 were shown to enhance
the GTPase activity of Mtb-FtsZ but inhibit the Mtb-FtsZ polymerization in a dose-dependent manner. 5f (SB-P17G-C2)
(54) and 7c-4 (SB-P17G-A20) (55)138 were identified as
potent bactericidal benzimidazoles in SAR studies. These
benzimidazoles did not show significant cytotoxicity against a
VERO eukaryotic cell line.137,138 Those potent benzimidazoles
effectively inhibited the polymerization of Mtb-FtsZ and also
caused the depolymerization of existing Mtb-Ftz protofilaments.138 52 and 55140,141 showed efficacy in acute tuberculosis
model studies in mice. Optimized analogs of this series of
compounds, SB-P17G-A38 (56) and SB-P17G-A42 (57),142
were recently reported to have efficacy in a tuberculosis
infection animal model.
3.1.9. FtsZ Inhibitors That Incorporate Other Heterocyclic
Scaffolds. Quinuclidines 1 (58)143 and 12 (59)144 differ by a
hydroxyl or N-methylamino group, respectively. Both compounds inhibit growth of a variety of bacterial species and have
broad-spectrum activity. The quinuclidine core was suggested
to bind to the GTP pocket of FtsZ through docking models
of the M. jannaschii crystal structure (PDB code 1W5B).
Fluorophores such as DAPI (60) have been shown to inhibit
the GTPase activity of FtsZ but do not affect polymerization of
tubulin.145 4-Bromo-1H-indazole 12 (61)146 was designed from
the charged alkaloids 1 and chelerythrine (62). 61 showed
moderate antibacterial activity with an MIC of at least 128 μM
against any species of bacteria tested. 5,5-Bis-8-anilino-1naphthalenesulfonate (63) inhibits the binding of GTP to
FtsZ and is significantly affected by the concentration of
calcium ions present. The concentration of calcium ions also
induces conformational changes of FtsZ and might thus be the
more important effect to consider in modulating the activity of
this protein.108
3.2. Altering FtsZ Activity or Stability by Targeting
FtsZ Regulators. Membrane−protein and protein−protein
interactions are critical for assembly of the divisome (Figure 2A
and Figure 2B). As described in section 2, proteins that
modulate FtsZ synthesis, polymerization, activity, and turnover
are essential for ensuring the precise spatiotemporal regulation
of cytokinesis. In principle, many of these factors can be
explored as potential targets for the development of FtsZ
inhibitors (Table 2), and yet this area is largely unexplored.
In the next section we describe three examples of general,
conserved FtsZ regulators that can be explored as potential
indirect targets of FtsZ.
3.2.1. Altering FtsZ Activity by Disrupting the ZipA−FtsZ
Interaction. In E. coli and other γ-proteobacteria, the transmembrane protein ZipA is one of the essential components of
the divisome responsible for recruitment of FtsZ to the
membrane (Table 2).36,50,69 ZipA binds specifically to residues
confined to the C-terminal region of FtsZ.36 There are at least
two examples in the literature of groups of small molecules that
disrupt the interactions between ZipA and FtsZ. The indolo[2,3a]quinolizin-7-one inhibitors (compounds 1 (64) and 10b (65))
and selected for its antibacterial potency and selectivity for
bacteria over mammalian cells. 31 was hypothesized to bind to
the putative colchicine pocket of M. tuberculosis FtsZ based on
chemoinformatics modeling. Rhodanines (e.g., OTBA (32))
increase protofilament assembly/bundling and inhibit GTPase
activity of FtsZ but do not affect the secondary structure of
FtsZ. 32, not surprisingly, also inhibits the proliferation of
HeLa cells118 and generally affects many proteins nonselectively, as evidenced by its proliferation as a “PAINs compound”
in high-throughput screens (vida infra). Aminofurazan A189
(33)119 was identified in a chromosome partitioning screen by
an anucleate cell blue assay that looked specifically for the
inhibition of GTPase activity of FtsZ, although no binding site
was predicted for the compound with either E. coli or S. aureus
FtsZ. Aminopyridines SRI-3072 (34) and SRI-7614 (35)120
inhibit the GTPase activity of M. tuberculosis FtsZ and reduced
bacterial growth in mouse bone marrow macrophages. UCM44
(36)121 inhibits the GTPase activity of FtsZ in B. subtilis but
only marginally for E. coli FtsZ.
A family of structurally unrelated small molecules referred to
as the “zantrins” were reported to affect FtsZ protofilament
assembly and inhibit GTPase activity; zantrins Z1 (37), Z2
(38), and Z4 (39) decreased the length of FtsZ protofilaments,
and zantrins Z3 (40) and Z5 (41) stabilized FtsZ protofilaments.122 37 and its chemical relative trisphenol 7 were further
pursued due to their shared structural scaffolds but were found
to be small molecules aggregators and not bona fide inhibitors
of FtsZ. Due to their poor prospects as drug leads, 38, 39, and
41 were not examined in subsequent studies. Recent SAR studies
of 40 demonstrated that a substituted quinazoline ring could
retain the potency of the parent compound and incorporation of a
small, positively charged side chain improved activity by 3-fold.123
3.1.7. FtsZ Inhibitors Based on a Benzamide Scaffold.
Studies of the benzamide family of small molecules over the
past 15 years culminated in the development of PC190723
(42)124 from the starting inhibitor 3-methoxybenzamide
(43).125 42 was the first non-nucleotide inhibitor of FtsZ to
be cocrystallized with FtsZ.126 42 was initially described as
stabilizing FtsZ protofilaments127 and inhibiting GTPase
activity;124 however two independent groups later demonstrated that the compound decreases the cooperativity of
FtsZ monomers,130 activates the GTPase activity of S. aureus
FtsZ,97,128 and resensitizes MRSA to β-lactams.126 The
cocrystal structure of FtsZ and 42 is consistent with binding
of this molecule between strand 8 and helix H7, which disrupts
the conserved hydrogen bonds that enable helix H7 to
communicate with the GTP binding site. This translocation
of helix H7 decreases the lag phase of FtsZ polymerization,
altering the cooperativity of the FtsZ monomers.126,128 42
mislocalizes the Z-ring in S. aureus cells; however it does not
disrupt the division proteins that localize to the FtsZ foci.129
Although limited to S. aureus, 42 is currently the best inhibitor
of FtsZ to date and is a useful tool for microbiology. However,
poor solubility and formulation properties have hindered 42
from clinical use. 42 has been modified into various prodrugs
such as TXY436 (44)130 to improve its poor oral bioavailability.
44 and 42 have been further developed into the metabolically
more stable analog TXA709 (45), in which the chlorine substituent is replaced with a trifluoromethyl group;131 preclinical
studies of this compound are in progress.132 Related benzamide
8J (46)129 is structurally similar to 42 and varies only at the
benzothiazole ring system, while (R)-13 (47)133 and compound
1 (48)134 contain the 3-alkoxy-2,6-difluorobenzamide core.
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division by binding to and sequestering monomeric FtsZ and
reducing the effective concentration of FtsZ in cells.169 SulA is
highly conserved among Enterobacteriaceae,170 and its role as a
potent inhibitor of bacterial cell division could be exploited as a
potential target for the development of antibiotics that inhibit
cell division by modulating FtsZ.
3.3. Inhibiting FtsZ by Disrupting the Cellular
Transmembrane Potential in Bacteria. Membrane potential (ΔΨ) is essential for proper subcellular localization of
some cell-division-related proteins, such as MinD and FtsA in
B. subtilis and E. coli.171 Consistent with this model, ionophores
(e.g., CCCP and valinomycin) and bacteriocins (e.g., nisin and
colicin N) that cause depolarization of the cell membrane
abolish the oscillation of MinD and the mid-cell localization of
FtsA. The detailed mechanism underlying ΔΨ-dependent
localization of these membrane proteins is not completely
understood, but in vitro experiments suggest that, at least for
MinD, the membrane potential stimulates the interaction
between the C-terminal amphipathic helix of MinD and the
phospholipid bilayer.171 A reduction in mid-cell localization of
FtsZ and ZapA occurs after treating cells with CCCP and is
correlated with FtsA mislocalization,171 which is important for
Z-ring stabilization at the membrane. This finding confirms that
inhibition of other proteins or cellular components that interact
with FtsZ and regulate FtsZ dynamics can be explored as
potential targets for altering FtsZ activity.
Many FtsZ inhibitors including 11, 1, 20, 9, and 37 affect the
oscillatory behavior of MinD by reducing the membrane
potential and affecting membrane permeability.172 As discussed
in section 4.6.1, some of the compounds classified as FtsZ
inhibitors in vitro do not cause cell filamentation, one of the
phenotypic hallmarks of FtsZ inhibition in vivo. These
observations suggest that the activity of these compounds on
FtsZ may arise as the downstream consequence of their effect
on bacterial membranes.
3.4. Inhibition of FtsZ Synthesis Using Short
Antisense Oligoribonucleotides. The finding that the
endonuclease ribonuclease P, essential for maturation of the
5′ end of tRNAs, can be used to digest target RNA molecules
upon addition of an appropriate complementary oligoribonucleotide led to the development of EGS technology.173 The
ability to interfere with f tsZ gene expression has been recently
investigated as an alternative therapeutic strategy to block
bacterial cell division.174 Expression of an EGS targeting the
f tsZ mRNA induces cell filamentation and causes growth
inhibition in E. coli cells. EGS techniques are still at an early
stage of development; however they have been used as antibacterial agents and to inhibit expression of resistance genes in
bacteria.175 In principle, EGS approaches could be an efficient
strategy to overcome the increase of multiresistance among
bacterial pathogens.
were shown to affect ZipA−FtsZ interaction by occupying a
hydrophobic cavity on the surface of ZipA necessary for the
binding to FtsZ. Consistent with this model, in vitro studies
showed that various analogs were able to inhibit binding of
ZipA to a small peptide that mimics the C-terminal 16 amino
acid residues of E. coli FtsZ.147 Similarly, a structure-based study
of carboxybiphenylindole inhibitors (e.g., compound 14 (66))
demonstrated binding of a small peptide that mimics the
C-terminal 16 amino acid residues of FtsZ and C-terminal
domain (residues 185−328) of ZipA.148
3.2.2. Modulating FtsZ Stability through Degradation by
the ClpXP Protease. ClpXP is a two-component ATPdependent bacterial protease that controls protein turnover
by proteolysis.149 The substrate recognition domain of ClpXP
(the ClpX chaperone) can function in a ClpP-independent
manner preventing protein assembly and aggregation or
remodeling and disassembling macromolecular complexes/
aggregates.150−152 In E. coli and in B. subtilis, the ClpX
chaperone inhibits formation of the Z-ring in a ClpPindependent fashion through a mechanism that does not
require hydrolysis of ATP, suggesting that ClpX physically
blocks the assembly of FtsZ protofilaments.153−155 Similarly,
ClpX regulates Z-ring assembly in M. tuberculosis by interacting with FtsZ. Consistent with the model, overexpression
of clpX inhibits Z-ring assembly and reduces viability of
M. tuberculosis.156
Genetic and biochemical studies in E. coli have shown that
the two-component ClpXP protease modulates the dynamics of
FtsZ filaments via degradation of FtsZ monomers and protofilaments.38 Bacterial cells overproducing ClpX or ClpXP arrest
cell division and have a filamentous morphology.38,153,155 These
observations demonstrate that the possible specific activation of
the proteolytic activity of ClpXP affecting the stability of FtsZ
could be an avenue for FtsZ inhibition and potential antibacterial agent development.
A new approach to inhibit cell division through FtsZ by
targeting the bacterial proteolytic machinery was demonstrated
recently.157−159 Acyldepsipeptides (ADEPs, 67−71) are natural
product-derived antibiotics active against Gram-positive bacteria, and their mechanism of action involves uncontrolled
proteolysis of FtsZ mediated by ClpP peptidase.159 Biochemical
and structural data showed that this family of compounds
competes with the Clp ATPases for the same binding site,
stimulates ClpP activity through cooperative binding, and
induces uncontrolled ClpP-dependent proteolysis, decreasing
the abundance of FtsZ and inhibiting cell division.157−160 It is
not clear why FtsZ is particularly sensitive to ADEP-ClpP;
however the mechanism is dependent on the structure of the
protein, as α- and β-tubulins are also targets of the ADEP-ClpP
complex.159
3.2.3. Inhibiting FtsZ by Activating the SOS Pathway.
Following DNA damage, E. coli and related Gram-negative
bacteria activate an elaborate cellular program (the SOS
response) for DNA repair and cell survival.161 The division
inhibitor SulA is synthesized as part of the SOS response,162,163
which causes cell filamentation by inhibiting polymerization
of FtsZ at the division site.164−166 After DNA is repaired, SulAmediated inhibition of FtsZ is rapidly released by proteolysis
of SulA,167 restoring the ability of FtsZ to polymerize and
re-form the Z-ring. SulA binds FtsZ monomers in a 1:1 ratio,
and GTP is required for SulA binding in vitro.165,168 Although
there is evidence that SulA inhibits the GTPase activity of
FtsZ,165,168 a recent study suggests that SulA inhibits cell
4. STRATEGIES FOR DISCOVERING NEW FtsZ
INHIBITORS
4.1. High-Throughput Screening. Target-based (largely
in vitro) and whole-cell (in vivo) high-throughput screens have
been used to identify FtsZ small molecule inhibitors. The
majority of target-based approaches require recombinant,
purified protein and do not select for compounds that have
favorable transport properties across bacterial membranes.176
The most common screens of FtsZ inhibitors have assayed
inhibition of the GTPase activity or FtsZ polymerization in the
presence of a small molecule.118,122,135 Other in vitro screens
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were not discovered in screens per se. Examples include 11,101
15,105 16,106 2,184 1,88 4,92 and 20111 (Table 3). Several of these
compounds have off-target effects185 and challenges with
aggregation.97
4.3. Virtual Screening Using the FtsZ Crystal
Structure. A virtual screening approach utilizes known
structural data of a prioritized target and identifies or optimizes
a ligand for a binding site of interest. Ligands can be chemical
structures extracted from databases or built from chemical
fragments docked into regions of the binding site.186 Databases
are constructed to include chemical structures with a high
degree of druglikeness with physically relevant conformations.187
Virtual screening using crystal structures of FtsZ have
identified new inhibitors. Specifically, inhibitors of the GTPase
activity of FtsZ have been predicted by docking compounds
into the GTP binding site of FtsZ. One virtual screen focused
on docking compounds from natural product collections,144
while another screen utilized known scaffolds from the
literature, an in-house synthetic library of small molecules,
and 4 000 000 compounds from a virtual library.121 Both of
these approaches identified compounds that are structurally
distinct from GTP and had moderate activity against FtsZ
in vitro. A select number of these compounds demonstrated
moderate antibacterial activity against Gram-positive organisms,121 confirming the value of this discovery approach.
4.4. Molecular Modeling and SAR Studies To Identify
FtsZ Inhibitors. Molecular modeling using a protein crystal
structure is often used to optimize hits from a high-throughput
screen to improve binding and activity. This strategy decreases
the cost and amount of time for SAR studies and provides
compounds for biochemical studies and further protein
crystallization. A new pharmocophore that inhibits the
interaction between FtsA and ZipA was identified in a highthroughput screen and cocrystallized with FtsZ.178,179 By use of
the shape-comparison program ROCS, new scaffolds were
identified that have potential for synthetic optimization.188
Rational design using molecular modeling is one possible
strategy when a protein crystal structure is available and
docking experiments can be performed.
For example, rational design of FtsZ inhibitors using
molecular modeling methods can leverage molecules that
mimic GTP, such as 27, compete with GTP for binding to
FtsZ, and inhibit the GTPase activity (Table 3). An analysis of
GTP bound to FtsZ in the crystal structure reveals positions on
the nucleotide that can be modified to obstruct the binding of
GTP to FtsZ.115 Docking experiments with 2 (Table 3) and
FtsZ predicted that 2 binds to the C-terminal cleft near the
GTP binding site and identified the C9-methoxy position of 2
as a possible locus for modification to optimize its activity.91
Another approach to inhibit FtsZ function is to use modeling to
target and disrupt the FtsZ interface with proteins that localize
it and affect its function. For example, docking experiments
were used to optimize the structure of 66 to disrupt the ZipA−
FtsZ interaction148 (Table 3).
SAR studies can also lead to potent analogs without
structural or computational guidance. By use of combinatorial
chemistry, GTP analogs with two side chain substitutions
(compounds 28, 29, and 30) in the place of the ribose and the
triphosphate were synthesized and tested for inhibition of the
GTPase activity of FtsZ116 (Table 3). An amine modification
library of 64 (an inhibitor from the study mentioned
previously148) resulted in a moderate increase in the inhibition
have measured the small molecule inhibition of the interaction
between FtsZ and ZipA using fluorescence polarization.177,178 A
challenge with in vitro assays of recombinant proteins is that
they often yield inhibitors that are not very active in vivo.
Challenges with cell uptake and bioavailability (e.g., drug efflux)
of inhibitors developed through in vitro high-throughput
approaches have created new momentum for whole-cell
chemical screens.176
Most whole-cell, high-throughput assays have been designed
to identify compounds that inhibit cell growth rather than to
focus on a specific antibacterial target. This strategy
serendipitously led to the identification of the quinolone and
quinazoline families of FtsZ inhibitors.117 Whole-cell strategies
have been developed to identify FtsZ inhibitors.121,174,175,179
A clever example of a cell-based reporter assay for FtsZ utilized
the activation of the σF transcription factor during sporulation
in B. subtilis as a reporter of septum formation at the site of cell
division. This dual reporter system monitors the expression
levels of promoter-fusions for the σF-dependent spoIIQ
promoter and spoIIA promoter, which is only active before
septation. In the presence of a cell division inhibitor, the
concentration of spoIIQ decreases and spoIIA increases, which
inhibits septum formation. The assay has a built-in control to
reduce nonspecific inhibitors, which cause the levels of spoIIQ
and spoIIA to decrease.180 Once a hit is identified in whole-cell
assays, secondary assays are used to confirm FtsZ as the target.
A growing consensus is that engineering cell permeability into a
lead compound identified from a screen is more difficult than
developing in vivo assays, and consequently whole-cell assays
are being used frequently for high-throughput screening.176
4.2. Screening Natural Product Libraries and Extracts
for FtsZ Inhibitors. Libraries of synthetic small molecules
have been widely assayed in high-throughput screens to identify
FtsZ inhibitors. An increasing number of screens have focused
on natural products. Natural products were the primary source
of antimicrobial compounds in the early years of antibiotic
discovery and comprise 77% of the antibiotics used clinically
since 2000 (all of which were derived from microbes).181
A decrease in the rate of antibiotic discovery, among other
factors, shifted attention from natural products to semisynthetic
natural products and entirely synthetic compounds. One key
challenge of antibiotic discovery is that the most successful
antibiotics often do not follow Lipinski’s rules of druglike
compounds. Consequently, libraries of synthetic compounds
(which are curated for compounds that follow Lipinski’s rules)
are missing key chemical space for antibiotic drug discovery.176
Natural products are intrinsically biologically active, and compounds have been evolutionarily selected for their transport
properties into bacterial cells.182 These and other properties
could fill this missing chemical space found in chemical
libraries. The development and maintenance of natural product
extract libraries have emerged as a method of increasing the
chemical diversity of compound libraries for antibiotic drug
discovery.183
High-throughput assays have been used to screen natural
product extract libraries for FtsZ inhibitors in vitro.93,96 Also,
virtual structural libraries of natural products and their
semisynthetic derivatives have been implemented using in
silico high-throughput assays.144 Preliminary whole-cell antibacterial activity screening of natural extracts from different
sources, such as marine fungi,109 marine alga,98 or certain pine
trees,112 have provided new chemical scaffolds that target FtsZ.
Many natural products identified as FtsZ inhibitors in vitro
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of the interaction between FtsZ and ZipA.147 The discovery of
42 is a successful SAR story that did not rely on in silico
strategies (Table 3). By use of 43 as a starting point, 500
analogs were synthesized and tested against bacteria, using cell
shape to assess biological activity.124 Substitutions on the
phenyl ring established that fluorine atoms at the R4 and R7
positions improved antibacterial activity. Compounds with alkyl
chains of various lengths at the 3-methoxy position were
>10 000 times more potent than 43.189 Further SAR studies
improved the druglike ADME properties of the 2,6-difluoro-3alkyloxybenzamides. The replacement of the alkyl chains with
heterocycles, such as thiazolopyridines and benzothiazoles,
decreased the log P and potential plasma protein binding. The
benzothiazole derivative retained its antibacterial activity while
lowering the log P value compared to the alkyloxybenzamide
derivatives. Analogs with substitutions at each of the available
positions on the benzothiazole ring were prepared and tested.
Incorporating a chlorine atom at the 5-position and replacing a
nitrogen atom at the 7-position of the benzothiazole produced
a low log P value, decreased plasma protein binding, and
retained potent antibacterial activity against S. aureus.190 Not
only does this example demonstrate a successful SAR study of
an FtsZ inhibitor, but it also highlights a constant goal in
medicinal chemistry to exploit “ligand efficiency.” This term has
emerged to describe the level of activity of an inhibitor on a per
atom or per dalton basis, which provides a metric for measuring
improved potency without sacrificing druglikeness.191
4.5. Screening and Modification of Tubulin Inhibitors.
Many potent and selective inhibitors of tubulin originate from
natural products and have gone on to become clinical drugs and
or drug leads, including 24, vinca alkaloids (vinblastine,
vincristine), 17, epothilone, peloruside, maytansine, and
halichondrin (the progenitor of eribulin/halaven). Structural
biology data have been determined for most of these protein−
small molecule complexes.192−195 The contrast of inhibitors of
tubulin and FtsZ is stark: there are far fewer natural products
that inhibit the function of FtsZ, none of these inhibitors have
the potency of the tubulin-targeting molecules listed above, and
only one cocrystal structure has been solved to date (FtsZ
bound to the synthetic small molecule 42). The origin of this
difference may reflect a research bias (e.g., a larger allocation of
federal funding to support eukaryotic cell biology), a difference
in the susceptibility of each protein to small molecule binders,
and/or differences in resistance mechanisms in eukaryotes versus
prokaryotes.
Vinca alkaloids and taxanes are drugs used for clinical cancer
chemotherapy by either destabilizing (vinca alkaloids) or
stabilizing microtubules (taxanes). Both activities prevent
normal microtubule function in cells. Some vinca alkaloids
are FDA-approved (such as vinblastine, vincristine, and
vinorelbine) for treating specific types of cancer including
lymphomas, sarcoma, leukemias, and non-small-cell lung
cancers. FDA-approved taxanes include docetaxel, 24, and
nab-24 for treating different types of cancer including breast,
gastric, head and neck, prostate, ovarian, and non-small-cell
lung cancers. Derivatives of epothilones are currently in clinical
development because of their increased potency compared to
taxanes and their application in combination drug therapy.196
Tubulin has been a validated cancer treatment target for many
years and is structurally similar to FtsZ, as we mentioned briefly
in section 2.1 and highlight in Figure 1.
Phylogenetic analyses reinforce an evolutionary linkage
between FtsZ and eukaryotic tubulin.13,32,197 In fact, FtsZ and
tubulin share many essential functional and structural properties, including cooperative assembly stimulated by GTP and
dynamic polymerization. The structural homology of Sus scrofa
tubulin and M. jannaschii FtsZ is high, and the common core is
superimposable with a root-mean-square deviation of 4.3 Å
(Figure 1).13 Despite excellent structural homology, distinctions between the two proteins are significant enough that they
may be exploited to create chemical inhibitors specific for FtsZ.
Loops connecting strands and helices of tubulin are longer
than those in FtsZ, causing tubulin to have a wider crosssection than a molecule of FtsZ. A sequence alignment of
tubulin and FtsZ demonstrates that the proteins share 7%
homology of amino acids, most of which are located in regions
associated with nucleotide binding.13 After polymerization in
vitro, FtsZ protofilaments associate laterally, which is different
from the association of tubulin protofilaments in microtubules
(Figure 1). Sheets of FtsZ protofilaments do not have a
standard tubulin microtubule lattice. Another physical distinction between the two proteins is that FtsZ minirings
consisting of protofilaments are approximately half the diameter
of tubulin rings.41 Although the two proteins are structurally
homologous, the protofilament bundling arrangement and
amino acid sequence of tubulin and FtsZ are distinct, which
provides a platform for target specificity.
A challenge of FtsZ inhibitor discovery is to identify
molecules that do not target eukaryotic tubulin, a step referred
to as the “antitubulin approach”. Many of the classic tubulin
inhibitors do not have significant activity against GTPase
activity and polymerization of FtsZ, demonstrating that target
specificity is possible. Examples of this specificity are the tubulin
inhibitors 17 and 51, both of which show no significant activity
against FtsZ polymerization and GTPase activity.16 Similarly,
the cross-species activity of an inhibitor can in principle be finetuned to target only FtsZ.
34 and 35 were identified as M. tuberculosis FtsZ inhibitors
from a synthetic tubulin inhibitor library120 (Table 3). An SAR
study enhanced the antibacterial activity of the compounds and
increased the specificity for inhibition of FtsZ over tubulin.198
The benzimidazole scaffold was picked out of the same tubulin
inhibitor library as a promising antagonist of FtsZ (Table 3),
and a library of 2,5,6- and 2,5,7-trisubstituted benzimidazoles
were synthesized and tested against drug-sensitive and drugresistant M. tuberculosis. A cyclohexyl group at the 2-position
was preferred and the results motivated the investigation of
substitutions at the 5- and 6-position of the benzimidazoles,137
which led to two potent analogs with a dimethylamino group
at the 6-position and a benzamide or a carbamate at the
5-position.138 Additionally, the investigation of the 6-position
was expanded to create a library of trisubstituted benzimidazoles
with ether and thioether substituents at the 6-position; however
none of the 6-ether/thioether analogs were as potent as the
analogs with the dimethylamino group at the 6-position.199
This SAR study is an excellent example of rational drug design
using tubulin inhibitors as a starting point to discover new
molecules with specificity for inhibiting FtsZ.
4.6. Strategies To Improve Characterization Methods
of FtsZ Inhibitors. Chemical hits against FtsZ from in vivo
whole-cell screens or natural product extracts are typically
confirmed using in vitro experiments, such as GTP hydrolysis
and protofilament assembly. Conversely, hits from in vitro highthroughput or virtual screens are tested in vivo for phenotypic
traits of FtsZ inhibitors, such as cell filamentation or subcellular
localization of the Z-ring.24
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A drug whose dose−response curve deviates from the standard
sigmoidal shape and instead forms a bell-shaped curve at higher
concentrations of drug should be scrutinized as a potential
aggregating molecule.205 One method for detecting the
presence of drug aggregates is implementing a detergentbased control using Triton X-100, which dissolves drug−drug
aggregates.206 However, Triton X-100 has been shown to
disrupt the protein−protein interactions between the tubulin
polymers as evidenced by electron microscopy.121 Nevertheless,
a cross-comparison of Triton X-100 versus centrifugation or
increasing protein concentration showed similar results for 37,
42, and 20.97 Furthermore, Triton X-100 has been used in control experiments with derivatives of 24113 and analogs of 2.91
Bona fide inhibitors of FtsZ will produce the same dose−
response curve with or without an aggregation test, while an
aggregator will result in a loss of activity at any drug concentration.
In vitro measurements of GTP hydrolysis by FtsZ are
complicated by the complexity of the biological coupling
between GTPase activity and FtsZ protofilament formation.
A thorough reexamination of several reported FtsZ inhibitors
indicates two are aggregators (e.g., 7 and 37); one is a PAINs
compound (e.g., 32), and several inhibitors have activity that
vary significantly from earlier reports (e.g., 9).97 Aggregation is
a problem for in vitro assays, such as GTPase activity
measurements and light scattering experiments, in which a
nonspecific aggregation effect of small molecule inhibitors with
FtsZ monomers impedes polymerization. FtsZ protofilaments
and bundles can be observed by electron microscopy; however
this technique is unable to decouple whether a decreasing FtsZ
polymerization rate and the change in the number of protofilaments are a result of aggregation or an inhibitor binding to
FtsZ specifically. FRET assays can be used to transcend this
limitation. One population of FtsZ proteins can be labeled with
a donor fluorophore (a FRET donor) and another with an
acceptor fluorophore (a FRET acceptor). Polymerization of
FtsZ monomers creates a spatial distribution of fluorophores; a
donor and acceptor positioned adjacent to each other will
produce a FRET signal.207 An inhibitor that disrupts FtsZ
polymerization may reduce the FRET signal.
Cell filamentation arises in response to alterations in the
topological state or structure of DNA. As mentioned in section
3.2.3, when the SOS response is triggered in response to DNA
damage, SulA inhibits FtsZ polymerization (Table 2) and
cell filamentation occurs. However, bacteria can filament via
SulA-independent mechanisms after the SOS response is
triggered by DNA damage, perturbation of DNA topology,
and stalling of the DNA replication fork.185,208 The first step in
the SOS response is controlled by the RecA-activated selfcleavage of LexA, a transcriptional repressor of all the SOS
response genes.208 Therefore, the use of a ΔsulA mutant with a
noncleavable LexA repressor (lexA (ind−)) is more informative
than a ΔsulA mutant, as the activation of all of the SOS
response genes and their contribution to SulA-independent
filamentation should be assessed.
As referred above in section 2.2, the depletion of other
Fts proteins yields filamentous cells that are multinucleate
(i.e., they have multiple copies of chromosomes that are evenly
spaced along the length of the filament),209 suggesting that
inhibiting Fts proteins (other than FtsZ) produces a filamentous
phenotype. Inhibiting some clinically relevant antibacterial
targets (such as inhibitors of peptidoglycan biosynthesis and
DNA supercoiling) produces a filamentous cell phenotype.
The in vitro measurement of GTP hydrolysis activity has
become a standard assay for FtsZ inhibitors. As mentioned
earlier, target-based high-throughput screens were designed to
monitor GTPase activity by measuring the release of inorganic
phosphate using a malachite green dye assay99,106,111,118,135,137
or coupled enzyme assays.122,200 A common secondary assay to
evaluate compounds in vitro is monitoring the inhibition or stabilization of FtsZ protofilaments and bundles using light scattering
or electron microscopy.88,89,101,105,106,115,118,127,135,137,179,201
Further in vitro studies can be performed to characterize
FtsZ inhibition by evaluating the intrinsic properties of FtsZ.
The conformational changes of FtsZ and its bundles in the
presence of inhibitor are measurable by far-UV circular
dichroism.100,105,106,118
Many assays have been developed to demonstrate compounds binding to FtsZ directly. A simple method is to perform
a sedimentation assay that takes advantage of the formation of
insoluble protofilaments as FtsZ polymerizes, which precipitate
out of solution and can be collected by centrifugation. If an
inhibitor prevents polymerization, the amount of sedimented
FtsZ decreases; conversely, the amount of sedimented FtsZ
increases if the inhibitor stabilizes protofilaments.111,118,122,127,135,179
Several examples of competitive binding assays have been
reported that use fluorescent inhibitors,106,108 fluorescent
probes,111 fluorescent GTP nonhydrolyzable analogs,99,111,179
and modification of FtsZ with fluorescein96 or tryptophan
residues.92,118,179 All of these in vitro techniques facilitate
characterizing compounds as GTPase inhibitors or activators
that disrupt or stabilize FtsZ protofilaments.
Microscopy strategies are commonly used to characterize
FtsZ inhibitors in vivo. Disruption of the Z-ring causes an
inhibition of cell division, thus giving the iconic filamentous cell
phenotype observed using microscopy.4,125 Further microscopy
studies have utilized ΔsulA mutants to determine that the
filamentation phenotypes are SulA-independent.88,96,122,184
By use of epifluorescence microscopy techniques, the mislocalization of the Z-ring can be visualized using an anti-FtsZ
antibody88,92,106,111,118,122,179 or functional fluorescently tagged
FtsZ.99,101,129,184 Additionally, the coupling of a chromosome
fluorophore (such as 60) with Z-ring localization experiments
establishes whether an inhibitor alters chromosome segregation.88,92,106,111,118,122,129,179 In summary, FtsZ inhibitors are
currently characterized in vivo as having a SulA-independent
filamentation phenotype with a mislocalized FtsZ ring and no
alteration of chromosome segregation.
4.6.1. Challenges Associated with Different Characterization Techniques for FtsZ Inhibitors. A challenge in the
search for bacterial cell division inhibitors is differentiating
“signal” (i.e., bona fide inhibitors) from “noise”(i.e., nonspecific
binders or compounds that target other aspects of the cell and
are translated into alterations in FtsZ activity). A variety of
different stimuli can trigger FtsZ inhibition, including
accumulation of drug aggregates, induction of DNA damage,
changes in transmembrane potential, and targeting the activity
of proteins positioned upstream of FtsZ, all of which may
present the sought-after filamentation phenotype of whole-cell
screens. Beyond the PAIN compounds that increasingly turn
out “false positives” in bioassays and screening libraries,202,203
druglike molecules can also give spurious results due to
nonspecific interactions of drug aggregates with proteins or
promiscuous drug−protein interactions.97,204 These aggregates
arise from the drug molecules forming organic, often hydrophobic particles in the aqueous environment of a bioassay.
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development; however many aspects of this regulatory network
remain enigmatic. Its central position in cell division highlights
FtsZ as a prime candidate for chemotherapeutic strategies, and
yet after nearly 2 decades of research on FtsZ inhibitors no
inhibitors have emerged for clinical use.
The plethora of papers on FtsZ antagonists and lack of
multiple, potent inhibitors of FtsZ suggest that finding small
molecules to target this protein is challenging. When the
activity of these molecules is considered in the context of the
many potent inhibitors of tubulin that have been clinically
developed, the medicinal chemistry of FtsZ appears to still be
stuck in very early stages of development and the field is faced
with an important question: Why does this large discovery
bandgap exist?
One possible explanation is that FtsZ is a much harder
protein to drug than tubulin. Comparisons of the crystal structures indicate that tubulin contains numerous regions for
binding small molecules, while FtsZ has fewer regions in which
molecules can bind, thereby reducing the probability of finding
an inhibitor that binds FtsZ antagonistically. Another related
explanation is that there is still very little crystal structure data
on FtsZ compared to tubulin. Although there are >30 crystal
structures deposited in PDB, ∼30% of them are of S. aureus
FtsZ and only one has a non-nucleotide small molecule bound
in the structure, 42, which provides limited information on how
compounds can alter FtsZ structure and activity. 42 binds to
S. aureus FtsZ in a flat orientation and appears to affect
polymerization by shifting the H7 helix marginally. However,
the general lack of crystallographic data makes it difficult to
learn how to design better inhibitors using structural biology
data. Additional structural biology data of FtsZ bound to the
most potent inhibitors could provide insight into design
principles; however the lack of available potent FtsZ inhibitors
limits this approach.
A second possibility centers upon current chemical libraries,
which do not often include new molecular entities that contain
structural features that are a hallmark of successful clinical
antibiotics. Commercial molecular libraries remain locked in a
mindset of Lipinski’s rules, which has been successful for
identifying inhibitors of individual proteins and compounds
active in eukaryotic cells (e.g., mammalian cells) but is not
particularly effective for targeting bacteria. Other sets of rules
for molecular properties that improve bacterial uptake (e.g.,
Moser’s rules)212 may be a more helpful tool for curating small
molecule libraries for families of molecules that may have
antibacterial properties. Specifically, these molecules may have
improved transport properties across bacterial membranes and
may be poor substrates for transport out of cells through efflux
pumps. Limitations of chemical space in synthetic chemical
libraries would benefit from following other druglikeness rules
for antibiotic drug development.
One way to fill this void in the chemical space of commercial
libraries is to refocus on natural products and libraries that
contain secondary metabolites; here dereplication and metagenomic techniques may be particularly effective. One
challenge with FtsZ however remains that it is widely conserved
among bacteria, which reduces the possibility that bacteria
evolved secondary metabolites to inhibit FtsZ in other cells. For
this mechanism to be possible, antibiotic-producing bacteria
would require a chaperone that keeps the inhibitor bound until
it is secreted. Alternatively, bacteria may evolve mutated forms
of FtsZ that have reduced binding to secondary metabolites
that they secrete to inhibit the growth of competing bacteria.
Consequently, validating that FtsZ inhibitors do not target
penicillin-binding proteins or DNA gyrase is an important step
in characterizing new drugs.210,211 Surprisingly, some FtsZ
inhibitors, such as the zantrins, compounds 37−41, and
hemi-10, do not filament cells (or do only very minimally),
yet they mislocalize the Z-ring.122 These observations lead to
questions surrounding whether FtsZ is the primary target of
these compounds.
Additionally, the mislocalization of the Z-ring can be
attributed to many factors that precede its formation. Positive
protein regulators of FtsZ are involved in localizing FtsZ to the
center of the cell (Table 2). Consequently, their inhibition
could result in a misplacement of the Z-ring. As discussed
previously in section 3.3, the localization of cytoskeletal and cell
division proteins has been shown to be sensitive to membrane
depolarization. The mislocalization of the Z-ring can also
be attributed to compromised membranes and a disrupted
membrane potential.171 Therefore, the characterization of a
possible FtsZ inhibitor should be accompanied by determining
whether it affects the membrane.
4.6.2. Strategies To Improve Screening and Characterization Approaches for New FtsZ Inhibitors. To improve the
chances of identifying new FtsZ inhibitors, new screening
methods should explore new genetic approaches and in vivo
techniques. In vivo high-throughput screens with clever cellbased reporter systems provide information about antibacterial
potency. Using overexpression strains of FtsZ or an enzyme
that interacts with it (Table 2) would distinguish small
molecule inhibitors whose antibacterial activity is reversed by
overexpression of these enzymes. In accordance with protein−
protein interactions, a small molecule activator of SulA (“fake”
DNA damage), ClpXP (proteolytic activity), or SlmA (pseudo
chromosome mis-segregation) would theoretically cause an
indirect stalling of the Z-ring.
Many recent detection techniques utilizing new instruments
could be implemented for in vivo screens. Microscopes and
flow cytometers have been used for high-throughput screening
by incorporating stage attachments and detectors that facilitate
the use of 96-well plates. Using a 96-well plate microscopy
assay, one could observe the cell filamentation and mislocalization of the Z-ring on a large scale that employs more individual
compound treatments than are possibly using serial, one-at-atime assays. Flow cytometers can simultaneously measure many
aspects of cell physiology (i.e., the length of cells) and the
intensity of a chosen fluorescent probe (i.e., 60) in seconds.
An experiment could entail grouping all of the in vivo characteristics of a small molecule into one flow cytometry
experiment using a ΔsulA lexA (ind−) mutant to control for
SOS-dependent cell filamentation, a fluorescently tagged FtsZ
to observe Z-ring mislocalization, and a quantifiable DNA stain
(such as Picogreen) to quantify the number of chromosomes
per nucleoid. The translation of biophysical assays from other
areas of biology could be useful for screening small molecule
libraries to identify new inhibitors of FtsZ.
5. SUMMARY AND FUTURE DIRECTIONS
Bacteria use a variety of regulatory mechanisms to influence the
initiation and progression of cell division, many of which
ultimately hinge on the essential cell division protein FtsZ.
These mechanisms ensure spatiotemporal coordination of cell
division and other biological processes necessary for cell
viability. Recent studies have uncovered new biochemistry
related to cell division that can be exploited for antibiotic
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Biographies
However, the lack of divergence among FtsZ indicates that it
may be difficult to mutate the protein and retain its enzymatic
function, assembly into filaments, force generation, and
contacts with other proteins. These characteristics may have
prevented genetic drift of FtsZ and dampened the development
of mechanisms of chemical warfare for inhibiting cell division
through targeting FtsZ. These considerations reduce the
feasibility of secondary metabolites that evolved to target
FtsZ. Perhaps some of the exciting new techniques for
secondary metabolite identification213 will turn up new
compounds with activity against FtsZ and motivate the field
to dig deeper into natural products.
One of the lessons learned through an analysis of FtsZ
inhibitors is the value of in-depth mechanism of action studies
to confirm on-target binding and rule out indirect mechanisms
of FtsZ antagonism (e.g., triggering the SOS pathway,
aggregation and promiscuous binding to proteins, and altering
cell membrane potential). Cell filamentation and Z-ring
mislocalization are characteristic phenotypes of FtsZ inhibitors;
however they can be caused by many different mechanisms.
FtsZ studies reveal an aspect of medicinal chemistry that is
important for drug design and development: the role of
structural biology in confirming and characterizing binding
interactions to the target. The lack of small molecules that have
been solved in cocrystal structures to FtsZ indicates either a
very limited repertoire of current drugs that are bona fide
inhibitors of FtsZ or an intrinsic challenge of crystallizing FtsZ
and solving the structure with a non-natural compound bound.
Targeting cell division with small molecules can leverage
many of the different biochemical steps (Table 2) that are
connected to FtsZ and cytokinesis. There are a variety of in
vivo assays that can be used (or new methods that can be
engineered) to qualitatively or quantitatively measure the
inhibition of cell division and are compatible with highthroughput screening methods. Bacterial cell division remains
an active area of fundamental research, and inhibitors of specific
proteins that participate in this process may be important tools
for studying the biochemical and biophysical mechanisms that
are involved. Although 42 has become the canonical FtsZ
inhibitor, it is only effective against S. aureus FtsZ and, for the
reasons highlighted earlier, it has provided limited understanding of how to design molecules to target this protein.
New families of clinical antibiotics against Gram-negative
bacteria that display multidrug resistance214,215 may motivate
the discovery of cell division inhibitors against these organisms.
FtsZ remains an attractive target for inhibiting division in
Gram-negative bacteria. Potent antagonists may have a dual use
in understanding how bacteria coordinate the multiple steps of
cell division and as antimicrobial agents, which may lead to new
clinical antibiotics for chemotherapies.
■
Katherine A. Hurley received her B.S. in Chemistry with a
pharmaceutical emphasis at University of CaliforniaDavis, CA, and
completed research studies under the supervision of Professor Jared T.
Shaw. She is currently pursuing a Ph.D. degree in Pharmaceutical
Sciences from the School of Pharmacy at the University of
WisconsinMadison, WI, under the supervision of Douglas Weibel.
Her present research involves discovering and characterizing new
antibiotics as chemotherapeutic agents and chemical biology probes.
Thiago M. A. Santos obtained his B.S. in Biological Sciences and a
M.S. degree in Agricultural Microbiology from Universidade Federal
de Viçosa, Brazil, and pursued predoctoral studies in the College of
Veterinary Medicine at Cornell University, NY. He is currently
pursuing a Ph.D. degree in the Microbiology Doctoral Training
Program at the University of WisconsinMadison, WI, under the
supervision of Douglas Weibel. His current research deciphers the
molecular mechanisms of protein localization in bacteria and the mode
of action of novel antimicrobial drugs.
Gabriella M. Nepomuceno received her B.S. in Chemistry at
University of CaliforniaSanta Cruz, CA, and completed research
studies under the supervision of Professor Bakthan Singaram. She
received a Ph.D. degree in Chemistry from the University of
CaliforniaDavis, CA, under the supervision of Jared T. Shaw. Her
research involved designing and synthesizing molecular probes in
chemical biology and organic methodology.
Valerie Huynh received her B.S. in Pharmaceutical Chemistry from
the University of CaliforniaDavis, CA, under the supervision of
Jared T. Shaw. She received a M.S. degree in Pharmaceutical
Chemistry at the same institution. Her research involved synthesizing
molecular probes for medicinal chemistry. Currently, she is a Research
Associate at Gilead Sciences, Inc.
Jared T. Shaw received his Ph.D. in Chemistry from Keith Woerpel at
University of CaliforniaIrvine and then moved to Harvard
University, MA, as an NIH Postdoctoral Fellow with David Evans.
He became an Institute Fellow at the Institute for Chemistry and Cell
Biology (ICCB) at Harvard Medical School where he helped found
the Center for Chemical Methodology and Library Development
(CMLD), which later became part of the Broad Institute of Harvard
and Massachusetts Institute of Technology. He is currently an
Associate Professor of Chemistry at the University of California
Davis, CA, and he currently works on the development of new
methods for the synthesis of natural products and other complex
molecules that modulate biological phenomena.
Douglas B. Weibel received his B.S. degree in Chemistry from the
University of Utah, was a Fulbright Fellow at Tohoku University,
Japan (with Yoshinori Yamamoto), and received his Ph.D. in
Chemistry from Cornell University, NY (with Jerrold Meinwald).
He was a Postdoctoral Fellow at Harvard University, MA (with
George Whitesides). He is currently an Associate Professor of
Biochemistry, Chemistry, and Biomedical Engineering at the
University of WisconsinMadison, WI, and his research spans the
fields of biochemistry, biophysics, chemistry, materials science and
engineering, and microbiology.
AUTHOR INFORMATION
Corresponding Authors
*J.T.S.: e-mail, [email protected]; phone, +1 (530) 752-9979.
*D.B.W.: e-mail, [email protected]; phone, +1 (608)
890-1342; fax, +1 (608) 265-0764.
■
ACKNOWLEDGMENTS
Due to space constraints, we were unable to cite all of the
research on FtsZ; any occlusions were unintentional. Research on
antibiotics in the Weibel laboratory has been supported by the
Human Frontiers Science Program (Grant RGY0076/2013), the
NIH (Grant 1DP2OD008735), the Wisconsin Alumni Research
Author Contributions
#
K.A.H. and T.M.A.S. contributed equally to this article.
Notes
The authors declare no competing financial interest.
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Foundation, and the USDA (Grant WIS01594). Research on
inhibitors of cell division in the Shaw laboratory is supported by
NIH/NIAID (Grants R01A108093, R01A08093-04S1).
■
ABBREVIATIONS USED
ANS, 8-anilinonaphthalene-1-sulfonic acid; ADME, absorption,
distribution, metabolism, and excretion; CCCP, carbonyl
cyanide m-chlorophenyl hydrazone; DAPI, 4′,6-diamidino-2phenylindole; EGS, external guide sequence; FDA, Food and
Drug Administration; FRAP, fluorescence recovery after
photobleaching; FRET, fluorescence resonance energy transfer;
GDP, guanosine diphosphate; GTP, guanosine triphosphate;
pN, piconewton; OTBA, 3-{5-[4-oxo-2-thioxo-3-(3trifluoromethylphenyl)thiazolidin-5-ylidenemethyl]furan-2-yl}benzoic acid; PAIN, pan-assay interference; ROCS, rapid
overlay of chemical structures; SAR, structure−activity relationship; UV, ultraviolet
■
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