Targeting the Bacterial Division Protein FtsZ - UW-Madison · Targeting the Bacterial Division...

Targeting the Bacterial Division Protein FtsZKatherine A. Hurley,†,# Thiago M. A. Santos,‡,# Gabriella M. Nepomuceno,§ Valerie Huynh,§

Jared T. Shaw,*,§ and Douglas B. Weibel*,‡,∥,⊥

†Department of Pharmaceutical Sciences, University of WisconsinMadison, 777 Highland Avenue, Madison, Wisconsin 53705,United States‡Department of Biochemistry, University of WisconsinMadison, 440 Henry Mall, Madison, Wisconsin 53706, United States§Department of Chemistry, University of CaliforniaDavis, One Shields Avenue, Davis, California 95616, United States∥Department of Chemistry, University of WisconsinMadison, 1101 University Avenue, Madison, Wisconsin 53706, United States⊥Department of Biomedical Engineering, University of WisconsinMadison, 1550 Engineering Drive, Madison, Wisconsin 53706,United States

ABSTRACT: Similar to its eukaryotic counterpart, the prokaryotic cytoskeleton is essential for the structural and mechanicalproperties of bacterial cells. The essential protein FtsZ is a central player in the cytoskeletal family, forms a cytokinetic ringat mid-cell, and recruits the division machinery to orchestrate cell division. Cells depleted of or lacking functional FtsZ donot divide and grow into long filaments that eventually lyse. FtsZ has been studied extensively as a target for antibacterialdevelopment. In this Perspective, we review the structural and biochemical properties of FtsZ, its role in cell biochemistry andphysiology, the different mechanisms of inhibiting FtsZ, small molecule antagonists (including some misconceptions aboutmechanisms of action), and their discovery strategies. This collective information will inform chemists on different aspects ofFtsZ that can be (and have been) used to develop successful strategies for devising new families of cell division inhibitors.

1. INTRODUCTION: TARGETING THE BACTERIALPROTEIN FtsZ

An increase of multidrug resistance to antibiotics amongpathogenic strains of bacteria and the lack of innovation inthe discovery of new antibacterial agents punctuate the needfor new chemotherapeutic strategies. One approach to newstrategies is the identification, characterization, and explorationof new molecular targets for antibiotic development, which iscurrently in vogue. Historically, all known clinical antibioticstarget one of the following bacterial structures and cellularprocesses: (1) DNA replication; (2) transcription; (3) transla-tion; (4) peptidoglycan biosynthesis; (5) folate biosynthesis;(6) the cytoplasmic membrane.1,2 An important, unansweredquestion is whether additional classes of mechanisms andtargets exist for developing new families of antibiotics. Thebacterial cytoskeleton is one such family of targets for whichclinical 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 essentialfor the physiological and structural properties of cells, includingcell 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, thebacterial cytoskeleton has been a topic of discussion for thedevelopment of antibacterial compounds over the past 2 decades.

The essential cytoskeletal cell division protein FtsZ (namedafter the filamenting temperature-sensitive mutant Z) is anessential GTPase structurally related to eukaryotic tubulins11−13

and highly conserved in bacteria and archaea.14,15 During celldivision, FtsZ forms a ringlike structure at the site of division andfunctions 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 aprime target for antibacterial development.16 The use of FtsZ asan antibacterial drug target has been reviewed,17,18 and itsstructural biology16,19,20 and inhibition with small moleculeshave been discussed.21−25 Specifically, targeting FtsZ with smallmolecules as a defense against tuberculosis has also beenextensively reviewed.26−28 In this review, we explore the latestdevelopments of classes of small molecules and inhibitorstargeting FtsZ and evaluate the challenges and future directionsof this field of antibiotic research.

2. STRUCTURE AND FUNCTION OF FtsZ

2.1. FtsZ Structure. FtsZ shares 40−50% sequence identityacross most bacterial and archaeal species and has a three-dimensional structure that is similar to the structure of α- and

Received: July 14, 2015Published: January 12, 2016

Perspective

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© 2016 American Chemical Society 6975 DOI: 10.1021/acs.jmedchem.5b01098J. Med. Chem. 2016, 59, 6975−6998

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http://dx.doi.org/10.1021/acs.jmedchem.5b01098

β-tubulin3,13,29,30 (Figure 1). Despite structural and functionalsimilarities, FtsZ is a distant ancestral homolog of tubulin withan amino acid sequence that is <20% identical.3,13,31,32

Crystallographic analysis of FtsZ from the hyperthermophilicmethanogen Methanocaldococcus jannaschii (formerly Methano-coccus jannaschii) revealed the presence of two domains

Table 1. Examples of Key Components of the Bacterial Cytoskeletona

cytoskeletal proteinb function/remarks

Tubulin-like

FtsZ -Is the structural subunit of the Z-ring

-Recruits downstream proteins involved in cell division and cell wall synthesis and remodeling

TubZ -Involved in DNA segregation

Actin-like

FtsA -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

MreB -Required for cell shape determination (morphogenesis) and maintenance

-Is also implicated in chromosome segregation and cell polarity

ParM -Participates in DNA segregation

Intermediate Filaments

crescentin -Responsible for the asymmetric cell shape in some bacteria (e.g., it is an essential determinant of the curved shapes of C. crescentus cells)

Walker A “Cytoskeletal” ATPases

MinD -Involved in positioning the Z-ring at mid-cell

ParA -Participates in DNA segregationaThis is not a comprehensive list. The bacterial cytoskeleton consists of other families of proteins or protein homologues that are absent from thislist. 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 andβ-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 andS. scrofa (PBD code 1TUB).30 The direction of the polymerization is indicated based on the axis of the protofilament. Bottom left: A representationof 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) boundin the active site. Each monomer is represented as a different shade of green to facilitate visualization, and GDP is represented as electrostatic spheresin brick red. Bottom right: A demonstration of the dimerization of one monomer of β-tubulin (dark green) with GDP bound in the active site andone monomer of α-tubulin (light green) with GTP bound in the active site from S. scrofa (PBD code 1TUB).30 The direction of the polymerizationis indicated based on the axis of the protofilament. These representations were generated using PyMOL (version 1.5.0.4).

Journal of Medicinal Chemistry Perspective

DOI: 10.1021/acs.jmedchem.5b01098J. Med. Chem. 2016, 59, 6975−6998

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connected by a long central helix (H7)13 (also designated as helixH529) (Figure 1). The amino-terminal portion of the proteinconsists of a six-stranded β-sheet sandwiched by two helices onone side and three on the other and contains the GTPase domain.This domain is conserved between FtsZ and tubulin; however, it isdifferent in other classic GTPases.13 The carboxy-terminal domainconsists of a four-stranded β-sheet in contact with helix H7 andsupported by two helices on one side while the other side isexposed to the solvent.29 The conserved C-terminal tail of FtsZmediates specific interactions between FtsZ and auxiliary proteinsthat 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 studiesin vivo demonstrate that the Escherichia coli Z-ring adopts a

compressed helical conformation with variable helical lengthand thickness of ∼110 nm.43 Electron cryotomographic recon-structions of dividing Caulobacter crescentus cells revealed thatthe Z-ring consists of short protofilaments that are ∼100 nmin length and randomly spaced near the division site andpositioned ∼16 nm away from the inner membrane.44 Morerecent electron cryomicroscopic and cryotomographic studiesin E. coli, C. crescentus, and constricting liposomes confirmedthe distance of the protofilaments positioned from the innermembrane and revealed that in both bacterial species the Z-ringis probably a continuous structure consisted of single-layeredbundles of FtsZ that are 5−10 filaments wide.45

2.2. Role of FtsZ in Cell Division. Bacterial cell divisionis a complex process that requires accurate identification ofthe division site, positioning of the division machinery, and

Figure 2. Spatiotemporal regulation of the Z-ring in different groups of bacteria. (A) Diagram summarizing the hierarchical recruitment of celldivision proteins in E. coli. FtsZ and the early cell division proteins localize to the division site before cell septation starts. The proteins are recruitedto the Z-ring in a sequential and approximately linear pathway. The requirement of an upstream protein for localization of a downstream protein tothe Z-ring was deduced from various studies of genetics, biochemistry, and microscopy. Proteins that regulate the assembly of the Z-ring are shownin green (positive regulators) and red (negative regulators). Peptidoglycan-specific amidases AmiA, AmiB, and AmiC play an important role incleaving the septum to release daughter cells after division in E. coli. AmiB and AmiC localize to the division site, whereas AmiA (not included in thediagram) is diffusely localized in the periplasm.216 This diagram was redrawn from refs 48, 51, and 217. (B) A cartoon illustrating some of thebacterial mechanisms for positioning of the Z-ring during cell division. In E. coli, Min proteins (MinCDE) oscillate between the cell poles, creating aninhibition zone (green shaded area) and preventing Z-ring (in red) polymerization near those poles.60,61,218 (For simplicity, the dynamic behavior ofthe Min system is omitted.) In addition, nucleoid occlusion, mediated by the protein SlmA, creates an inhibition zone (blue shaded area) along thecylindrical region of the cell and prevents Z-ring assembly over the nucleoid.63 Inset: A cartoon depicting the predicted organization of the E. colidivisome. Cell division is initiated with the polymerization of FtsZ into the Z-ring onto which the divisome apparatus assembles. The cartoon of thedivisome was adapted from refs 48 and 217. Similar to E. coli, the B. subtilis Min system (composed of MinCDJ and DivIVA) creates a zone ofinhibition (purple shaded area) that prevents Z-ring assembly at the cell poles. However, in B. subtilis, MinCDJ localizes to the cell poles in a DivIVA-dependent manner and does not undergo the characteristic dynamic oscillatory behavior observed in E. coli.219,220 In addition to the Min system, theprotein 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 andtemporal cues. The assembly of Z-ring is coincident with the subcellular position that exhibits the lowest concentration of MipZ. Prior tochromosome replication, MipZ and FtsZ are localized to the opposite poles of the cell. MipZ forms a complex with proteins involved in chromosomepartitioning. Following duplication of the chromosomal replication origin region (oriC), MipZ migrates toward the opposite cell pole creating abipolar gradient displacing the FtsZ at the poles and directing formation of the Z-ring toward mid-cell.65,221 In S. pneumoniae, the protein MapZforms 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.



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coordinated constriction of the inner membrane and the cellwall (i.e., cytokinesis). With few exceptions,46,47 this essentialprocess is initiated with the polymerization of FtsZ into afilamentous, ringlike structure (referred to as the Z-ring) that islocated in the cytoplasm peripheral to the membrane and closeto the division site.5,43−45 Concomitant with and following itspolymerization, the Z-ring recruits and coordinates a seriesof auxiliary proteins that perform diverse roles in cell divisionand cell wall biosynthesis and remodeling48−51 (Figure 2A).Depletion of FtsZ in rod-shaped bacteria, such as the Gram-negative E. coli, produces long, filamentous cells due to thecontinued growth of cells that are no longer dividing.4

Cocci-shaped bacteria, such as the Gram-positive pathogenStaphylococcus aureus, increase in volume up to 8-fold whendepleted of FtsZ.52 In both cases, cells are unable to divide;continued growth makes them enlarged and sensitive tochanges in the physical properties of their environment, andthe cells eventually lyse. Drugs that affect the positioning,activity, and interaction of FtsZ with other division proteinscause cell lysis and may be useful as antibiotics.2.3. FtsZ Dynamics during the Division Cycle. The

spatiotemporal regulation of Z-ring formation requires acomplex and concerted network of proteins that modulateassembly and activity of FtsZ to ensure that the division processis tightly coordinated with DNA replication, chromosomesegregation, and cell elongation.48,53−56 The molecular detailsunderlying the synchronicity of these processes are notcompletely understood; however, structural and cell biologyresearch over the past 2 decades has elucidated important struc-tural, functional, and regulatory aspects of these mechanismsand 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 celldivision proteins, as well as their regulators, are recruited tomid-cell in a hierarchical order to form the functional divisome,a ringlike multiprotein complex that constricts during the pro-cess of division and disappears when the cells separate49,50,57,59

(Figure 2B). The divisome machinery is essential and appearsto be widely conserved among bacteria.2.4. Regulatory Proteins That Position FtsZ in

Bacteria. Rod-shape bacteria (such as E. coli and the Gram-positive bacterium Bacillus subtilis) use at least two coordinatedbiochemical systems to accurately position the Z-ring at themid-cell and ensure that divisome formation is timed to occurat the final stage of the cell cycle: (1) the Min system ofproteins prevents aberrant division at regions other than themid-cell60,61 and (2) nucleoid occlusion proteins preventdivision from occurring over segregating chromosomes62,63

(Figure 2B). Importantly, in the absence of these two negativeregulators of Z-ring positioning, both E. coli and B. subtilis stillhave a bias for Z-ring formation at mid-cell,62,63 suggestingthat additional mechanisms may influence FtsZ assembly at themid-cell and coordinate chromosome segregation and celldivision. A recent study provided evidence of an additionalpositional marker in E. coli cells grown in minimal media andlacking functional Min and nucleoid occlusion systems.64 Inparticular, the authors identified that the Ter macrodomainregion of the chromosome acts as a landmark for the Z-ringin the presence of the chromosomal terminus organizationprotein MatP and the cell division proteins ZapA and ZapB.64

Much of the mechanistic insight on the spatiotemporalregulation of the Z-ring has come from studies of E. coli and

B. subtilis. However, many bacteria lack both canonical systemsfor positioning FtsZ. For example, in the Gram-negativebacterium C. crescentus, spatiotemporal assembly and placementof the Z-ring require MipZ.65 MipZ is an ATPase that asso-ciates with the origin region of chromosomes and (in a manneranalogous to the Min system) directly guides FtsZ positioningand polymerization into the Z-ring at mid-cell65 (Figure 2B).In the Gram-positive pathogen Streptococcus pneumoniae, MapZlocalizes at the division site prior to FtsZ, interacts directly withFtsZ, and guides positioning of the Z-ring66 (Figure 2B). MapZis a single-passage transmembrane protein that is conservedamong Streptococcaceae and other Lactobacillales and formsringlike structures positioned at mid-cell and at future divisionsites. In addition to its dual role in marking the cell division siteand positioning FtsZ, the balance between the phosphorylatedand dephosphorylated forms of MapZ may be important forcontrolling Z-ring stability and regulation of constriction.66

In addition to these specific protein-based systems thatcontrol FtsZ dynamics, other positive and negative regulatorsinteract with FtsZ to modulate structure and function of thedivisome in response to the nutritional and developmental stateof the cell (Table 2). These regulatory systems for positioningof FtsZ have adapted to different environments, cell shapes, anddevelopmental behaviors and emphasize the importance ofcoordinating the correct timing of cell division and chromosomesegregation. Many studies have indicated that unknownregulators of bacterial FtsZ may be awaiting discovery.

2.5. FtsZ Biophysics and Mechanics. An average E. colicell during log-phase growth contains ∼15 000 molecules ofFtsZ.67 The Z-rings assemble at the future site of division inE. coli daughter cells before the Z-ring is fully constricted in theparental cell.59 This observation suggests that future divisionsites in daughter cells become competent for assembly of thedivisome prior to the complete division of the mother cell. Inaddition, FRAP experiments in E. coli cells demonstrate that theZ-ring is a dynamic structure, continuously remodeled byexchanging subunits with the cytoplasmic pool of FtsZ (half-time of recovery ∼30 s). The kinetics of FtsZ turnover in vivo istightly coupled to GTP hydrolysis, as mutant cells with reducedGTPase activity of FtsZ show ∼9-fold slower turnover ofFtsZ into protofilaments.68 In vitro studies performed atphysiological conditions show that purified E. coli FtsZassembles into protofilaments and hydrolyze GTP at a rate of∼5 molecules per min per FtsZ, demonstrating that theGTPase activity of FtsZ in vitro is very slow.67

In addition to functioning as an essential molecular scaffoldfor recruitment and organization of the other cell divisionproteins to the division site,48,69 FtsZ generates a contractileforce that constricts tubular liposomes in vitro.70−72 FtsZ isthought to act as an important source of the constriction forcerequired for cytokinesis during cell division; however, themechanism by which FtsZ generates mechanical force andpromotes invagination of the cell wall during division remainsunclear. Previous studies with purified FtsZ have shown thatthe GTP-bound FtsZ assembles into straight or gently curvedfilaments, while the GDP-bound FtsZ forms highly curvedfilaments,41,42 suggesting that the difference in the intrinsiccurvature of FtsZ filaments provides a mechanism for gener-ating 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 issufficient to pull the cell wall inward at the division site toinitiate the constriction, and forces of 8−80 pN could lead to cell



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division by creating a reasonably accurate septal morphology.73

Molecular simulations of FtsZ dynamics estimate that FtsZ cangenerate a value of ∼20−30 pN per polymerized monomerwhen GTP is hydrolyzed,74 which is sufficient to direct cell−wall invagination during division and cause membrane vesicleformation from liposomes in vitro.70−72,75 Although mathe-matical models quantitatively support the “hydrolyze-and-bend”mechanism for force generation of the Z-ring,41,76,77 slightlycurved GTP-FtsZ filaments (without GTP-hydrolysis) arecapable of supplying a force of ∼10 pN, suggesting thatnucleotide hydrolysis might not be required for membranebending by FtsZ.74,75 This observation could explain theoccasional division events observed in cells containing a mutantversion of FtsZ that has very low GTPase activity78−80 and theinitial constriction of tubular liposomes in the presence of this“GTPase-dead” mutant FtsZ.75

2.6. FtsZ and Cell Morphology. The peptidoglycan layerof bacterial cell walls consists of a heteropolymer ofpolysaccharides cross-linked with short peptides that functionas the load-bearing material to resist mechanical and physicalforces (e.g., osmotic pressure) on cells. During the growth ofrod-shaped cells, peptidoglycan is assembled in two distinctregions of the cell: (1) along the cylindrical body of cells, whichis required for cell elongation, and (2) at the site of celldivision, which creates a new curved pole for the two daughtercells.FtsZ is required for septal/cell-division-associated peptido-

glycan growth and remodeling due to its essential role inrecruiting cell-division-specific peptidoglycan synthesis en-zymes.49,50 However, recent research suggests that the regu-latory role of FtsZ on peptidoglycan synthesis during cell

division extends beyond its ability to recruit proteins to themid-cell.7 Particularly, it was recently shown that the intrinsicallydisordered C-terminal linker region of FtsZ is important forregulation of enzymes involved in peptidoglycan metabolism inC. crescentus.7

In addition to septal/cell-division-associated peptidoglycangrowth mediated by the divisome, rod-shaped bacteria haveother cellular machinery mediating lateral peptidoglycansynthesis along the length of the cell. This multiproteincomplex named the “elongasome” is organized by the ancestralhomologue of actin, MreB.49 Until recently, the role of FtsZhas been thought to be restricted to participating in pepti-doglycan assembly and remodeling at the division site.However, recent studies have demonstrated that FtsZ mayalso play a role in elongation-associated cell wall growth in rod-shape bacteria.6,81,82

The direct role of an FtsZ homologue in cell shape control ofrod-shaped microorganisms has been also demonstrated inarchaea. Unlike most bacteria, archaeal genomes frequentlycontain additional genes belonging to the FtsZ/tubulinsuperfamily.83 The archaeal tubulin-like protein CetZ, formerlyannotated as “FtsZ3” or “FtsZ type 2”, has been implicated incell shape control of Haloferax volcanii.84 CetZ has the FtsZ/tubulin superfamily fold and a crystal form containing sheets ofprotofilaments 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-shapedcells into a rod-shaped cell type essential for normal swimmingmotility. CetZ1 forms dynamic cytoskeletal structures in vivo,indicating its capacity to remodel the cell envelope and directrod formation.84

Table 2. Proteins That Regulate the Formation of the Z-Ring in Bacteriaa

(1) Positive Regulators of Z-Ring Formation51,69

FtsAb -Membrane anchor supports assembly and stabilization of the Z-ring. It is also important for the recruitment of downstream proteins necessary for divisomematuration.

SepF -Required for proper morphology of the divisional septum. It has overlapping roles with FtsA in Z-ring assembly.

ZapA,B,C,D -Mediates additional stabilization of the Z-ring.

ZipA -Secondary membrane anchor that together with FtsA supports assembly of the Z-ring.

(2) Negative Regulators of Z-Ring Formation

ClpX/ClpXP -Helps modulate the equilibrium between the cytoplasmic pool of unassembled FtsZ and polymeric FtsZ through degradation.38 ClpX chaperone can also inhibitformation of the Z-ring in a ClpP-independent fashion by physically blocking the assembly of FtsZ filaments.153−155

CrgA -Important for coordinating cell growth and division. It regulates the dynamics of Z-ring formation and affects both the timing of FtsZ expression and itsturnover.224

EzrA -Modulates the position of the Z-ring during cell division and plays a role in coordinating cell growth and division.69

GdhZ -NAD-dependent glutamate dehydrogenase that controls Z-ring disassembly by stimulating the GTPase activity of FtsZ.225

KidO -Coordinates cellular or developmental activities with the availability of NADH. KidO bound to NADH is thought to destabilize lateral interactions betweenFtsZ protofilaments. It has been recently proposed to work in synergy with GdhZ to trigger Z-ring disassembly.225,226

OpgH -Glucosyltransferase that functions as a nutrient-dependent antagonist of the Z-ring. OpgH is thought to sequester FtsZ from growing polymers. Blocks Z-ringformation to coordinate cell growth and cell division.227

MciZ -Inhibits Z-ring formation by capping the minus end of FtsZ filaments and shortening the filaments.228

MinC -Important for positioning the Z-ring at mid-cell.60,61,218

MipZ -Required for positioning the Z-ring at mid-cell.65

MapZ -Important for Z-ring formation and positioning at mid-cell. It is also involved in the regulation of cytokinesis.66

Noc -Inhibits Z-ring formation over segregating chromosomes.62

SlmA -Analogous to Noc, it inhibits Z-ring formation over segregating chromosomes.63

SulA -Negative modulator of Z-ring expressed in response to DNA damage as part of the SOS system.165,168,169

UgtP -Similar to OpgH, this glucosyltransferase inhibits cell division by blocking Z-ring formation in a growth rate-dependent fashion. This cellular sensor ensures thatcells reach the appropriate mass and complete chromosome segregation prior to cytokinesis.229

YneA -Analogous to SulA, it regulates cell division through the suppression of Z-ring formation during the SOS response.230

aSome of these proteins and the molecular systems that they compose are widespread among bacteria. However, some of them are exclusive tospecific groups of bacteria. See the text and refer to the cited literature for an additional explanation. The referencing is not exhaustive for the best-studied proteins listed. bIt was recently demonstrated that FtsA has a dual, antagonistic role on the FtsZ filament network. FtsA is involved inrecruitment of FtsZ filaments to the membrane, but it also provides a negative regulation by causing fragmentation of FtsZ polymers, allowing therapid disassembly of FtsZ filaments.222



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3. MECHANISMS OF ACTION OF INHIBITORS OF FtsZ

Several characteristics validate FtsZ as a target for the devel-opment of new antibiotics to selectively combat bacterial infec-tions: (1) it is essential and plays a specific role in prokaryoticcell division;4 (2) it is structurally and functionally conservedacross bacterial and archaeal species;15,29,85 (3) althoughwidespread in mitochondria of diverse protist lineages, it isnotably absent in higher eukaryotes;83,86 (4) it is evolutionarilydistant 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 GTPrequires assembly of two FtsZ monomers to complete thecatalytic site. This innate step in catalysis can be modulated bytargeting either the T7 loop of the “upper” monomer or thenucleotide-binding pocket of the “lower” monomer.87 Alter-natively, an allosteric site on FtsZ may modulate its ability toform protofilaments. In this sense, the tightly regulated divisionprocess could be halted by several mechanisms, including(1) overly stabilizing protofilaments, which cannot disassembleas GDP is produced by GTP hydrolysis; (2) destabilizing proto-filaments; and (3) preventing polymerization. The chemicalinhibitors of FtsZ reported to date can be classified into threemain groups: (1) natural products and their derivatives;(2) nucleotide analogs; and (3) molecules that emerged fromhigh-throughput screening. Below, we provide an overview ofthe inhibitors that were a starting point for further developmentof structurally related compounds or assays for their activity asinhibitors of cell division. However, a caveat to this list is thatmany of these inhibitors have been shown to be false positivesor to have irreproducible activity. This section delineates thereported, albeit limited, mechanistic detail of reported FtsZinhibitors, which lays the foundation for a discussion of vali-dated inhibitors in section 4. A summary of the antimicrobialactivity, discovery methods, and FtsZ binding characteristics ofthese compounds is presented in Table 3.3.1.1. FtsZ Inhibitors from Natural Products: Alkaloids.

Sanguinarine (1) is a polycyclic alkaloid that inhibits FtsZprotofilament assembly by decreasing FtsZ polymerization;88 italso inhibits eukaryotic tubulin, which complicates its use as anantibiotic. Berberine (2) is a structurally related alkaloid thatinhibits GTPase activity and decreases FtsZ polymerization.It is predicted to bind in the vicinity of the GTP binding pocketand overlaps with several hydrophobic residues located in theGTP binding site.89 Although allegedly indifferent to tubulin, 2has since been described as a promiscuous binder of differentproteins.90 Berberine 2 (3) was designed to have an extendedalkyl group in place of one of the methyl groups on 2; the invitro GTPase inhibition activity of 3 was measured to beapproximately 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 in-creases the lag phase of FtsZ assembly (i.e., adversely affects thenucleation rate). The predicted binding site of 4 is located closeto 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 wassynthesized as an analog of 4 and shown to inhibit the GTPaseactivity of Bacillus anthracis slightly better than 2. Resveratrol (6)has been screened many times due to its known antimicrobialactivity, which has been attributed to inhibiting Z-ring formationand suppressing the expression of FtsZ mRNA.92,94

Dichamanetin (7) and 2‴-hydroxy-5″-benzylisouvarinol-B(8) are structurally similar pinocembrin-based molecules thatinhibit the GTPase activity of FtsZ in Gram-positive bacteria. 8also displays antimicrobial activity against E. coli andPseudomonas aeruginosa.95 However, 7 was later shown to bean “aggregator”, a molecule that forms aggregates that bindnonspecifically to proteins. Viriditoxin (9) was initially reportedto inhibit the GTPase activity of FtsZ and cause cells tofilament, while overexpression of FtsZ was shown to rescuedrug-treated cells.96 9 has since been confirmed as a false-positive that has activity that has not been reproducible.97 Thecomplex natural product family of chrysophaentins (e.g.,chrysophaentin A (10)) was shown to inhibit the GTPaseactivity of E. coli and S. aureus FtsZ (including methicillin-resistant S. aureus strains), and molecular docking experimentsshowed that the compound occludes a large portion of theGTP binding site of the protein.98 FtsZ polymerization isinhibited, and the Z-ring is mislocalized in cells treated with 10.Despite these results, cell filamentation was not observed in amutant strain of E. coli (envA1) permeable to a wide variety ofcompounds.99

3.1.3. FtsZ Inhibitors from Natural Products: Phenyl-propanoids and Terpenoids. Several phenylpropanoids thatare derived from cinnamaldehyde (11) or related structureshave been tested for antimicrobial activity. Nearly all phenyl-propanoids described as FtsZ inhibitors to date are alleged tointeract with at least one residue of the T7 loop.100 Virtualscreens and/or docking experiments of many of thesestructurally “simple” natural products suggest they have specificinteractions with FtsZ. However, few examples have translatedinto reliable inhibitors and lack biophysical support fortargeting FtsZ.11 inhibits the GTPase activity of FtsZ, decreases polymer-

ization and is not toxic to red blood cells.101 Phenylacrylamide14 (12)102 has antibacterial activity against S. aureus andStreptococcus pyogenes and inhibited cell division in S. aureus.Vanillin derivatives 3a (13)103 and 4u (14)104 have beenindependently tested against Mycobacterium tuberculosis FtsZ.Scopoletin (15), a coumarin analog related to esculetin andquercetin, inhibits the GTPase activity and polymerization ofFtsZ into protofilaments.105 Curcumin (16) increases theGTPase activity and destabilizes polymerization of FtsZ, thusreducing the steady-state duration of polymer assembly.106

Unlike other phenylpropanoids, the predicted FtsZ binding siteof 16 involves residues connected to GTP binding of FtsZ.107

Colchicine (17), although highly active against tubulin poly-merization, has also been tested against FtsZ and has beendemonstrated to have no effect on FtsZ polymerization.108

Sulfoalkylresorcinol (18) inhibits the GTPase activity ofFtsZ in vitro and exhibits antimicrobial activity against variouspathogens but is not cytotoxic toward human A549 cells.109

Synthetic derivative n-undecyl gallate (19) disrupts ZapAlocalization and possibly Z-ring formation in Xanthomonas citrisubsp. citri.110

Totarol (20) is a terpenoid that inhibits the GTPase activityand polymerization of FtsZ protofilaments. Cells treated with20 become filamentous and display a mislocalized Z-ring.111 20was initially described as lacking activity against eukaryotictubulin; more recently it has been shown to be promiscuous inbinding to proteins and to have properties consistent withbeing an aggregator.97 Germacrene D (21) and germacreneD-4-ol (22) are part of a family of terpenoids isolated from theessential 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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various species of bacteria. A docking model predicts a bindingsite of the germacrene family to be a hydrophobic pocket inFtsZ; however, the crystallographic evidence for this interactionis not yet determined.112

3.1.4. FtsZ Inhibitors That Are Derived from Taxanes.Taxane-derived structures have been successfully modified totarget FtsZ preferentially over its eukaryotic homologuetubulin. The taxane polycyclic core in these compounds hasremained largely intact. For example, SB-RA-2001 (23)113 onlydiffers from paclitaxel (24) in two ways: (1) the alcohol at C-10lacks an acetyl group, and (2) an unsaturated ester has replacedthe α-hydroxy-β-amido ester of 24 at C-13. These structuralchanges led to inhibition of enzymatic activity against B. subtilisFtsZ and were shown to have antimicrobial activity againstboth B. subtilis and Mycobacterium smegmatis. Huang et al. latershowed that the conjugated ester coupled with a ring-opened

core and altered oxidation pattern (denoted as TRA 10a (25)or 10b (26)) improved the potency of this taxane-derivedstructure to target M. tuberculosis FtsZ.114

3.1.5. FtsZ Inhibitors That Mimic Nucleosides. Nucleotideanalogs have been explored as competitive inhibitors of GTPfor binding to FtsZ. 8-Bromoguanosine 5′-triphosphate (27)binds to FtsZ with a Ki of 32 μM and inhibits both FtsZ poly-merization and GTPase activity.115 Gal cores 10 (28), 14 (29),and 15 (30)116 were designed to mimic the sugar-phosphatebackbone of GTP and shown to inhibit the GTPase activity ofP. aeruginosa FtsZ through an enzyme-coupled assay.

3.1.6. FtsZ Inhibitors from High-Throughput Screening ofChemical Libraries. A number of structurally distinct smallmolecules emerged from in vivo high-throughput screens ascausing cell filamentation and were considered to be targetingFtsZ. Quinoline 1 (31)117 was screened against M. tuberculosis

Table 3. continued

aEc = Escherichia coli; EcFtsZ = recombinant E. coli FtsZ; SaFtsZ = recombinant Staphylococcus aureus FtsZ; BsFtsZ = recombinant Bacillus subtilisFtsZ; MtFtZ = recombinant Mycobacterium tuberculosis FtsZ; PaFtsZ = recombinant Pseudomonas aeruginosa FtsZ; BaFtsZ = recombinant Bacillusanthracis FtsZ; MRSA = methicillin-resistant Staphylococcus aureus



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and selected for its antibacterial potency and selectivity forbacteria over mammalian cells. 31 was hypothesized to bind tothe putative colchicine pocket of M. tuberculosis FtsZ based onchemoinformatics modeling. Rhodanines (e.g., OTBA (32))increase protofilament assembly/bundling and inhibit GTPaseactivity of FtsZ but do not affect the secondary structure ofFtsZ. 32, not surprisingly, also inhibits the proliferation ofHeLa cells118 and generally affects many proteins nonselec-tively, 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 byan anucleate cell blue assay that looked specifically for theinhibition of GTPase activity of FtsZ, although no binding sitewas predicted for the compound with either E. coli or S. aureusFtsZ. Aminopyridines SRI-3072 (34) and SRI-7614 (35)120

inhibit the GTPase activity of M. tuberculosis FtsZ and reducedbacterial growth in mouse bone marrow macrophages. UCM44(36)121 inhibits the GTPase activity of FtsZ in B. subtilis butonly marginally for E. coli FtsZ.A family of structurally unrelated small molecules referred to

as the “zantrins” were reported to affect FtsZ protofilamentassembly 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 protofila-ments.122 37 and its chemical relative trisphenol 7 were furtherpursued due to their shared structural scaffolds but were foundto be small molecules aggregators and not bona fide inhibitorsof FtsZ. Due to their poor prospects as drug leads, 38, 39, and41 were not examined in subsequent studies. Recent SAR studiesof 40 demonstrated that a substituted quinazoline ring couldretain the potency of the parent compound and incorporation of asmall, 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 thepast 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 tobe cocrystallized with FtsZ.126 42 was initially described asstabilizing FtsZ protofilaments127 and inhibiting GTPaseactivity;124 however two independent groups later demon-strated that the compound decreases the cooperativity ofFtsZ monomers,130 activates the GTPase activity of S. aureusFtsZ,97,128 and resensitizes MRSA to β-lactams.126 Thecocrystal structure of FtsZ and 42 is consistent with bindingof this molecule between strand 8 and helix H7, which disruptsthe conserved hydrogen bonds that enable helix H7 tocommunicate with the GTP binding site. This translocationof helix H7 decreases the lag phase of FtsZ polymerization,altering the cooperativity of the FtsZ monomers.126,128 42mislocalizes the Z-ring in S. aureus cells; however it does notdisrupt the division proteins that localize to the FtsZ foci.129

Although limited to S. aureus, 42 is currently the best inhibitorof FtsZ to date and is a useful tool for microbiology. However,poor solubility and formulation properties have hindered 42from clinical use. 42 has been modified into various prodrugssuch as TXY436 (44)130 to improve its poor oral bioavailability.44 and 42 have been further developed into the metabolicallymore stable analog TXA709 (45), in which the chlorine sub-stituent is replaced with a trifluoromethyl group;131 preclinicalstudies of this compound are in progress.132 Related benzamide8J (46)129 is structurally similar to 42 and varies only at thebenzothiazole ring system, while (R)-13 (47)133 and compound1 (48)134 contain the 3-alkoxy-2,6-difluorobenzamide core.

Compound 297F (49)135 is the most structurally divergentcompound in the benzamide family of FtsZ inhibitors andtargets M. tuberculosis; however, it bears little structuralresemblance to 42.

3.1.8. FtsZ Inhibitors Based on a Benzimidazole Scaffold.A series of novel trisubstituted benzimidazoles, which wereinspired by tubulin-targeting thiabendazole (50) and albenda-zole (51),136 have been reported as targeting M. tuberculosisFtsZ (Mtb-FtsZ).137−142 Two benzaimidazoles, 1a-G7(SB-P3G2) (52) and 1a-G4 (53),137 were shown to enhancethe GTPase activity of Mtb-FtsZ but inhibit the Mtb-FtsZ poly-merization in a dose-dependent manner. 5f (SB-P17G-C2)(54) and 7c-4 (SB-P17G-A20) (55)138 were identified aspotent bactericidal benzimidazoles in SAR studies. Thesebenzimidazoles did not show significant cytotoxicity against aVERO eukaryotic cell line.137,138 Those potent benzimidazoleseffectively inhibited the polymerization of Mtb-FtsZ and alsocaused the depolymerization of existing Mtb-Ftz protofila-ments.138 52 and 55140,141 showed efficacy in acute tuberculosismodel studies in mice. Optimized analogs of this series ofcompounds, SB-P17G-A38 (56) and SB-P17G-A42 (57),142

were recently reported to have efficacy in a tuberculosisinfection animal model.

3.1.9. FtsZ Inhibitors That Incorporate Other HeterocyclicScaffolds. Quinuclidines 1 (58)143 and 12 (59)144 differ by ahydroxyl or N-methylamino group, respectively. Both com-pounds inhibit growth of a variety of bacterial species and havebroad-spectrum activity. The quinuclidine core was suggestedto bind to the GTP pocket of FtsZ through docking modelsof the M. jannaschii crystal structure (PDB code 1W5B).Fluorophores such as DAPI (60) have been shown to inhibitthe GTPase activity of FtsZ but do not affect polymerization oftubulin.145 4-Bromo-1H-indazole 12 (61)146 was designed fromthe charged alkaloids 1 and chelerythrine (62). 61 showedmoderate antibacterial activity with an MIC of at least 128 μMagainst any species of bacteria tested. 5,5-Bis-8-anilino-1-naphthalenesulfonate (63) inhibits the binding of GTP toFtsZ and is significantly affected by the concentration ofcalcium ions present. The concentration of calcium ions alsoinduces conformational changes of FtsZ and might thus be themore important effect to consider in modulating the activity ofthis protein.108

3.2. Altering FtsZ Activity or Stability by TargetingFtsZ Regulators. Membrane−protein and protein−proteininteractions are critical for assembly of the divisome (Figure 2Aand Figure 2B). As described in section 2, proteins thatmodulate FtsZ synthesis, polymerization, activity, and turnoverare essential for ensuring the precise spatiotemporal regulationof cytokinesis. In principle, many of these factors can beexplored as potential targets for the development of FtsZinhibitors (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 potentialindirect targets of FtsZ.

3.2.1. Altering FtsZ Activity by Disrupting the ZipA−FtsZInteraction. In E. coli and other γ-proteobacteria, the trans-membrane protein ZipA is one of the essential components ofthe divisome responsible for recruitment of FtsZ to themembrane (Table 2).36,50,69 ZipA binds specifically to residuesconfined to the C-terminal region of FtsZ.36 There are at leasttwo examples in the literature of groups of small molecules thatdisrupt the interactions between ZipA and FtsZ. The indolo[2,3-a]quinolizin-7-one inhibitors (compounds 1 (64) and 10b (65))



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were shown to affect ZipA−FtsZ interaction by occupying ahydrophobic cavity on the surface of ZipA necessary for thebinding to FtsZ. Consistent with this model, in vitro studiesshowed that various analogs were able to inhibit binding ofZipA to a small peptide that mimics the C-terminal 16 aminoacid residues of E. coli FtsZ.147 Similarly, a structure-based studyof carboxybiphenylindole inhibitors (e.g., compound 14 (66))demonstrated binding of a small peptide that mimics theC-terminal 16 amino acid residues of FtsZ and C-terminaldomain (residues 185−328) of ZipA.1483.2.2. Modulating FtsZ Stability through Degradation by

the ClpXP Protease. ClpXP is a two-component ATP-dependent bacterial protease that controls protein turnoverby proteolysis.149 The substrate recognition domain of ClpXP(the ClpX chaperone) can function in a ClpP-independentmanner preventing protein assembly and aggregation orremodeling and disassembling macromolecular complexes/aggregates.150−152 In E. coli and in B. subtilis, the ClpXchaperone inhibits formation of the Z-ring in a ClpP-independent fashion through a mechanism that does notrequire hydrolysis of ATP, suggesting that ClpX physicallyblocks the assembly of FtsZ protofilaments.153−155 Similarly,ClpX regulates Z-ring assembly in M. tuberculosis by inter-acting with FtsZ. Consistent with the model, overexpressionof clpX inhibits Z-ring assembly and reduces viability ofM. tuberculosis.156

Genetic and biochemical studies in E. coli have shown thatthe two-component ClpXP protease modulates the dynamics ofFtsZ filaments via degradation of FtsZ monomers and proto-filaments.38 Bacterial cells overproducing ClpX or ClpXP arrestcell division and have a filamentous morphology.38,153,155 Theseobservations demonstrate that the possible specific activation ofthe proteolytic activity of ClpXP affecting the stability of FtsZcould be an avenue for FtsZ inhibition and potential anti-bacterial agent development.A new approach to inhibit cell division through FtsZ by

targeting the bacterial proteolytic machinery was demonstratedrecently.157−159 Acyldepsipeptides (ADEPs, 67−71) are naturalproduct-derived antibiotics active against Gram-positive bac-teria, and their mechanism of action involves uncontrolledproteolysis of FtsZ mediated by ClpP peptidase.159 Biochemicaland structural data showed that this family of compoundscompetes with the Clp ATPases for the same binding site,stimulates ClpP activity through cooperative binding, andinduces uncontrolled ClpP-dependent proteolysis, decreasingthe abundance of FtsZ and inhibiting cell division.157−160 It isnot clear why FtsZ is particularly sensitive to ADEP-ClpP;however the mechanism is dependent on the structure of theprotein, as α- and β-tubulins are also targets of the ADEP-ClpPcomplex.159

3.2.3. Inhibiting FtsZ by Activating the SOS Pathway.Following DNA damage, E. coli and related Gram-negativebacteria activate an elaborate cellular program (the SOSresponse) for DNA repair and cell survival.161 The divisioninhibitor SulA is synthesized as part of the SOS response,162,163

which causes cell filamentation by inhibiting polymerizationof FtsZ at the division site.164−166 After DNA is repaired, SulA-mediated inhibition of FtsZ is rapidly released by proteolysisof SulA,167 restoring the ability of FtsZ to polymerize andre-form the Z-ring. SulA binds FtsZ monomers in a 1:1 ratio,and GTP is required for SulA binding in vitro.165,168 Althoughthere is evidence that SulA inhibits the GTPase activity ofFtsZ,165,168 a recent study suggests that SulA inhibits cell

division by binding to and sequestering monomeric FtsZ andreducing the effective concentration of FtsZ in cells.169 SulA ishighly conserved among Enterobacteriaceae,170 and its role as apotent inhibitor of bacterial cell division could be exploited as apotential target for the development of antibiotics that inhibitcell division by modulating FtsZ.

3.3. Inhibiting FtsZ by Disrupting the CellularTransmembrane Potential in Bacteria. Membrane poten-tial (ΔΨ) is essential for proper subcellular localization ofsome cell-division-related proteins, such as MinD and FtsA inB. subtilis and E. coli.171 Consistent with this model, ionophores(e.g., CCCP and valinomycin) and bacteriocins (e.g., nisin andcolicin N) that cause depolarization of the cell membraneabolish the oscillation of MinD and the mid-cell localization ofFtsA. The detailed mechanism underlying ΔΨ-dependentlocalization of these membrane proteins is not completelyunderstood, but in vitro experiments suggest that, at least forMinD, the membrane potential stimulates the interactionbetween the C-terminal amphipathic helix of MinD and thephospholipid bilayer.171 A reduction in mid-cell localization ofFtsZ and ZapA occurs after treating cells with CCCP and iscorrelated with FtsA mislocalization,171 which is important forZ-ring stabilization at the membrane. This finding confirms thatinhibition of other proteins or cellular components that interactwith FtsZ and regulate FtsZ dynamics can be explored aspotential targets for altering FtsZ activity.Many FtsZ inhibitors including 11, 1, 20, 9, and 37 affect the

oscillatory behavior of MinD by reducing the membranepotential and affecting membrane permeability.172 As discussedin section 4.6.1, some of the compounds classified as FtsZinhibitors in vitro do not cause cell filamentation, one of thephenotypic hallmarks of FtsZ inhibition in vivo. Theseobservations suggest that the activity of these compounds onFtsZ may arise as the downstream consequence of their effecton bacterial membranes.

3.4. Inhibition of FtsZ Synthesis Using ShortAntisense Oligoribonucleotides. The finding that theendonuclease ribonuclease P, essential for maturation of the5′ end of tRNAs, can be used to digest target RNA moleculesupon addition of an appropriate complementary oligoribonu-cleotide led to the development of EGS technology.173 Theability to interfere with f tsZ gene expression has been recentlyinvestigated as an alternative therapeutic strategy to blockbacterial cell division.174 Expression of an EGS targeting thef tsZ mRNA induces cell filamentation and causes growthinhibition in E. coli cells. EGS techniques are still at an earlystage of development; however they have been used as anti-bacterial agents and to inhibit expression of resistance genes inbacteria.175 In principle, EGS approaches could be an efficientstrategy to overcome the increase of multiresistance amongbacterial pathogens.

4. STRATEGIES FOR DISCOVERING NEW FtsZINHIBITORS4.1. High-Throughput Screening. Target-based (largely

in vitro) and whole-cell (in vivo) high-throughput screens havebeen used to identify FtsZ small molecule inhibitors. Themajority of target-based approaches require recombinant,purified protein and do not select for compounds that havefavorable transport properties across bacterial membranes.176

The most common screens of FtsZ inhibitors have assayedinhibition of the GTPase activity or FtsZ polymerization in thepresence of a small molecule.118,122,135 Other in vitro screens



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have measured the small molecule inhibition of the interactionbetween FtsZ and ZipA using fluorescence polarization.177,178 Achallenge with in vitro assays of recombinant proteins is thatthey 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-throughputapproaches have created new momentum for whole-cellchemical screens.176

Most whole-cell, high-throughput assays have been designedto identify compounds that inhibit cell growth rather than tofocus on a specific antibacterial target. This strategyserendipitously led to the identification of the quinolone andquinazoline families of FtsZ inhibitors.117 Whole-cell strategieshave been developed to identify FtsZ inhibitors.121,174,175,179

A clever example of a cell-based reporter assay for FtsZ utilizedthe activation of the σF transcription factor during sporulationin B. subtilis as a reporter of septum formation at the site of celldivision. This dual reporter system monitors the expressionlevels of promoter-fusions for the σF-dependent spoIIQpromoter and spoIIA promoter, which is only active beforeseptation. In the presence of a cell division inhibitor, theconcentration of spoIIQ decreases and spoIIA increases, whichinhibits septum formation. The assay has a built-in control toreduce nonspecific inhibitors, which cause the levels of spoIIQand spoIIA to decrease.180 Once a hit is identified in whole-cellassays, secondary assays are used to confirm FtsZ as the target.A growing consensus is that engineering cell permeability into alead compound identified from a screen is more difficult thandeveloping in vivo assays, and consequently whole-cell assaysare being used frequently for high-throughput screening.176

4.2. Screening Natural Product Libraries and Extractsfor FtsZ Inhibitors. Libraries of synthetic small moleculeshave been widely assayed in high-throughput screens to identifyFtsZ inhibitors. An increasing number of screens have focusedon natural products. Natural products were the primary sourceof antimicrobial compounds in the early years of antibioticdiscovery and comprise 77% of the antibiotics used clinicallysince 2000 (all of which were derived from microbes).181

A decrease in the rate of antibiotic discovery, among otherfactors, shifted attention from natural products to semisyntheticnatural products and entirely synthetic compounds. One keychallenge of antibiotic discovery is that the most successfulantibiotics often do not follow Lipinski’s rules of druglikecompounds. 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 com-pounds have been evolutionarily selected for their transportproperties into bacterial cells.182 These and other propertiescould fill this missing chemical space found in chemicallibraries. The development and maintenance of natural productextract libraries have emerged as a method of increasing thechemical diversity of compound libraries for antibiotic drugdiscovery.183

High-throughput assays have been used to screen naturalproduct extract libraries for FtsZ inhibitors in vitro.93,96 Also,virtual structural libraries of natural products and theirsemisynthetic derivatives have been implemented using insilico high-throughput assays.144 Preliminary whole-cell anti-bacterial activity screening of natural extracts from differentsources, such as marine fungi,109 marine alga,98 or certain pinetrees,112 have provided new chemical scaffolds that target FtsZ.Many natural products identified as FtsZ inhibitors in vitro

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 thesecompounds have off-target effects185 and challenges withaggregation.97

4.3. Virtual Screening Using the FtsZ CrystalStructure. A virtual screening approach utilizes knownstructural data of a prioritized target and identifies or optimizesa ligand for a binding site of interest. Ligands can be chemicalstructures extracted from databases or built from chemicalfragments docked into regions of the binding site.186 Databasesare constructed to include chemical structures with a highdegree of druglikeness with physically relevant conforma-tions.187

Virtual screening using crystal structures of FtsZ haveidentified new inhibitors. Specifically, inhibitors of the GTPaseactivity of FtsZ have been predicted by docking compoundsinto the GTP binding site of FtsZ. One virtual screen focusedon docking compounds from natural product collections,144

while another screen utilized known scaffolds from theliterature, an in-house synthetic library of small molecules,and 4 000 000 compounds from a virtual library.121 Both ofthese approaches identified compounds that are structurallydistinct from GTP and had moderate activity against FtsZin vitro. A select number of these compounds demonstratedmoderate antibacterial activity against Gram-positive organ-isms,121 confirming the value of this discovery approach.

4.4. Molecular Modeling and SAR Studies To IdentifyFtsZ Inhibitors. Molecular modeling using a protein crystalstructure is often used to optimize hits from a high-throughputscreen to improve binding and activity. This strategy decreasesthe cost and amount of time for SAR studies and providescompounds for biochemical studies and further proteincrystallization. A new pharmocophore that inhibits theinteraction between FtsA and ZipA was identified in a high-throughput screen and cocrystallized with FtsZ.178,179 By use ofthe shape-comparison program ROCS, new scaffolds wereidentified that have potential for synthetic optimization.188

Rational design using molecular modeling is one possiblestrategy when a protein crystal structure is available anddocking experiments can be performed.For example, rational design of FtsZ inhibitors using

molecular modeling methods can leverage molecules thatmimic GTP, such as 27, compete with GTP for binding toFtsZ, and inhibit the GTPase activity (Table 3). An analysis ofGTP bound to FtsZ in the crystal structure reveals positions onthe nucleotide that can be modified to obstruct the binding ofGTP to FtsZ.115 Docking experiments with 2 (Table 3) andFtsZ predicted that 2 binds to the C-terminal cleft near theGTP binding site and identified the C9-methoxy position of 2as a possible locus for modification to optimize its activity.91

Another approach to inhibit FtsZ function is to use modeling totarget and disrupt the FtsZ interface with proteins that localizeit and affect its function. For example, docking experimentswere 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 combinatorialchemistry, GTP analogs with two side chain substitutions(compounds 28, 29, and 30) in the place of the ribose and thetriphosphate were synthesized and tested for inhibition of theGTPase activity of FtsZ116 (Table 3). An amine modificationlibrary of 64 (an inhibitor from the study mentionedpreviously148) resulted in a moderate increase in the inhibition



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of the interaction between FtsZ and ZipA.147 The discovery of42 is a successful SAR story that did not rely on in silicostrategies (Table 3). By use of 43 as a starting point, 500analogs were synthesized and tested against bacteria, using cellshape to assess biological activity.124 Substitutions on thephenyl ring established that fluorine atoms at the R4 and R7

positions improved antibacterial activity. Compounds with alkylchains of various lengths at the 3-methoxy position were>10 000 times more potent than 43.189 Further SAR studiesimproved the druglike ADME properties of the 2,6-difluoro-3-alkyloxybenzamides. The replacement of the alkyl chains withheterocycles, such as thiazolopyridines and benzothiazoles,decreased the log P and potential plasma protein binding. Thebenzothiazole derivative retained its antibacterial activity whilelowering the log P value compared to the alkyloxybenzamidederivatives. Analogs with substitutions at each of the availablepositions on the benzothiazole ring were prepared and tested.Incorporating a chlorine atom at the 5-position and replacing anitrogen atom at the 7-position of the benzothiazole produceda low log P value, decreased plasma protein binding, andretained potent antibacterial activity against S. aureus.190 Notonly does this example demonstrate a successful SAR study ofan FtsZ inhibitor, but it also highlights a constant goal inmedicinal chemistry to exploit “ligand efficiency.” This term hasemerged to describe the level of activity of an inhibitor on a peratom or per dalton basis, which provides a metric for measuringimproved potency without sacrificing druglikeness.191

4.5. Screening and Modification of Tubulin Inhibitors.Many potent and selective inhibitors of tubulin originate fromnatural products and have gone on to become clinical drugs andor drug leads, including 24, vinca alkaloids (vinblastine,vincristine), 17, epothilone, peloruside, maytansine, andhalichondrin (the progenitor of eribulin/halaven). Structuralbiology data have been determined for most of these protein−small molecule complexes.192−195 The contrast of inhibitors oftubulin and FtsZ is stark: there are far fewer natural productsthat inhibit the function of FtsZ, none of these inhibitors havethe potency of the tubulin-targeting molecules listed above, andonly one cocrystal structure has been solved to date (FtsZbound to the synthetic small molecule 42). The origin of thisdifference may reflect a research bias (e.g., a larger allocation offederal funding to support eukaryotic cell biology), a differencein the susceptibility of each protein to small molecule binders,and/or differences in resistance mechanisms in eukaryotes versusprokaryotes.Vinca alkaloids and taxanes are drugs used for clinical cancer

chemotherapy by either destabilizing (vinca alkaloids) orstabilizing microtubules (taxanes). Both activities preventnormal microtubule function in cells. Some vinca alkaloidsare FDA-approved (such as vinblastine, vincristine, andvinorelbine) for treating specific types of cancer includinglymphomas, sarcoma, leukemias, and non-small-cell lungcancers. FDA-approved taxanes include docetaxel, 24, andnab-24 for treating different types of cancer including breast,gastric, head and neck, prostate, ovarian, and non-small-celllung cancers. Derivatives of epothilones are currently in clinicaldevelopment because of their increased potency compared totaxanes and their application in combination drug therapy.196

Tubulin has been a validated cancer treatment target for manyyears and is structurally similar to FtsZ, as we mentioned brieflyin 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 proper-ties, including cooperative assembly stimulated by GTP anddynamic polymerization. The structural homology of Sus scrofatubulin and M. jannaschii FtsZ is high, and the common core issuperimposable with a root-mean-square deviation of 4.3 Å(Figure 1).13 Despite excellent structural homology, distinc-tions between the two proteins are significant enough that theymay 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 cross-section than a molecule of FtsZ. A sequence alignment oftubulin and FtsZ demonstrates that the proteins share 7%homology of amino acids, most of which are located in regionsassociated with nucleotide binding.13 After polymerization invitro, FtsZ protofilaments associate laterally, which is differentfrom the association of tubulin protofilaments in microtubules(Figure 1). Sheets of FtsZ protofilaments do not have astandard tubulin microtubule lattice. Another physical dis-tinction between the two proteins is that FtsZ miniringsconsisting of protofilaments are approximately half the diameterof tubulin rings.41 Although the two proteins are structurallyhomologous, the protofilament bundling arrangement andamino acid sequence of tubulin and FtsZ are distinct, whichprovides a platform for target specificity.A challenge of FtsZ inhibitor discovery is to identify

molecules that do not target eukaryotic tubulin, a step referredto as the “antitubulin approach”. Many of the classic tubulininhibitors do not have significant activity against GTPaseactivity and polymerization of FtsZ, demonstrating that targetspecificity is possible. Examples of this specificity are the tubulininhibitors 17 and 51, both of which show no significant activityagainst FtsZ polymerization and GTPase activity.16 Similarly,the cross-species activity of an inhibitor can in principle be fine-tuned to target only FtsZ.34 and 35 were identified as M. tuberculosis FtsZ inhibitors

from a synthetic tubulin inhibitor library120 (Table 3). An SARstudy enhanced the antibacterial activity of the compounds andincreased the specificity for inhibition of FtsZ over tubulin.198

The benzimidazole scaffold was picked out of the same tubulininhibitor library as a promising antagonist of FtsZ (Table 3),and a library of 2,5,6- and 2,5,7-trisubstituted benzimidazoleswere synthesized and tested against drug-sensitive and drug-resistant M. tuberculosis. A cyclohexyl group at the 2-positionwas preferred and the results motivated the investigation ofsubstitutions at the 5- and 6-position of the benzimidazoles,137

which led to two potent analogs with a dimethylamino groupat the 6-position and a benzamide or a carbamate at the5-position.138 Additionally, the investigation of the 6-positionwas expanded to create a library of trisubstituted benzimidazoleswith ether and thioether substituents at the 6-position; howevernone of the 6-ether/thioether analogs were as potent as theanalogs with the dimethylamino group at the 6-position.199

This SAR study is an excellent example of rational drug designusing tubulin inhibitors as a starting point to discover newmolecules with specificity for inhibiting FtsZ.

4.6. Strategies To Improve Characterization Methodsof FtsZ Inhibitors. Chemical hits against FtsZ from in vivowhole-cell screens or natural product extracts are typicallyconfirmed using in vitro experiments, such as GTP hydrolysisand protofilament assembly. Conversely, hits from in vitro high-throughput or virtual screens are tested in vivo for phenotypictraits of FtsZ inhibitors, such as cell filamentation or subcellularlocalization of the Z-ring.24



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The in vitro measurement of GTP hydrolysis activity hasbecome a standard assay for FtsZ inhibitors. As mentionedearlier, target-based high-throughput screens were designed tomonitor GTPase activity by measuring the release of inorganicphosphate using a malachite green dye assay99,106,111,118,135,137

or coupled enzyme assays.122,200 A common secondary assay toevaluate compounds in vitro is monitoring the inhibition or stabili-zation of FtsZ protofilaments and bundles using light scatteringor electron microscopy.88,89,101,105,106,115,118,127,135,137,179,201

Further in vitro studies can be performed to characterizeFtsZ inhibition by evaluating the intrinsic properties of FtsZ.The conformational changes of FtsZ and its bundles in thepresence of inhibitor are measurable by far-UV circulardichroism.100,105,106,118

Many assays have been developed to demonstrate com-pounds binding to FtsZ directly. A simple method is to performa sedimentation assay that takes advantage of the formation ofinsoluble protofilaments as FtsZ polymerizes, which precipitateout of solution and can be collected by centrifugation. If aninhibitor prevents polymerization, the amount of sedimentedFtsZ decreases; conversely, the amount of sedimented FtsZincreases if the inhibitor stabilizes protofilaments.111,118,122,127,135,179

Several examples of competitive binding assays have beenreported that use fluorescent inhibitors,106,108 fluorescentprobes,111 fluorescent GTP nonhydrolyzable analogs,99,111,179

and modification of FtsZ with fluorescein96 or tryptophanresidues.92,118,179 All of these in vitro techniques facilitatecharacterizing compounds as GTPase inhibitors or activatorsthat disrupt or stabilize FtsZ protofilaments.Microscopy strategies are commonly used to characterize

FtsZ inhibitors in vivo. Disruption of the Z-ring causes aninhibition of cell division, thus giving the iconic filamentous cellphenotype observed using microscopy.4,125 Further microscopystudies have utilized ΔsulA mutants to determine that thefilamentation phenotypes are SulA-independent.88,96,122,184

By use of epifluorescence microscopy techniques, the mis-localization of the Z-ring can be visualized using an anti-FtsZantibody88,92,106,111,118,122,179 or functional fluorescently taggedFtsZ.99,101,129,184 Additionally, the coupling of a chromosomefluorophore (such as 60) with Z-ring localization experimentsestablishes whether an inhibitor alters chromosome segrega-tion.88,92,106,111,118,122,129,179 In summary, FtsZ inhibitors arecurrently characterized in vivo as having a SulA-independentfilamentation phenotype with a mislocalized FtsZ ring and noalteration of chromosome segregation.4.6.1. Challenges Associated with Different Character-

ization Techniques for FtsZ Inhibitors. A challenge in thesearch for bacterial cell division inhibitors is differentiating“signal” (i.e., bona fide inhibitors) from “noise”(i.e., nonspecificbinders or compounds that target other aspects of the cell andare translated into alterations in FtsZ activity). A variety ofdifferent stimuli can trigger FtsZ inhibition, includingaccumulation of drug aggregates, induction of DNA damage,changes in transmembrane potential, and targeting the activityof proteins positioned upstream of FtsZ, all of which maypresent the sought-after filamentation phenotype of whole-cellscreens. Beyond the PAIN compounds that increasingly turnout “false positives” in bioassays and screening libraries,202,203

druglike molecules can also give spurious results due tononspecific interactions of drug aggregates with proteins orpromiscuous drug−protein interactions.97,204 These aggregatesarise from the drug molecules forming organic, often hydro-phobic particles in the aqueous environment of a bioassay.

A drug whose dose−response curve deviates from the standardsigmoidal shape and instead forms a bell-shaped curve at higherconcentrations of drug should be scrutinized as a potentialaggregating molecule.205 One method for detecting thepresence of drug aggregates is implementing a detergent-based control using Triton X-100, which dissolves drug−drugaggregates.206 However, Triton X-100 has been shown todisrupt the protein−protein interactions between the tubulinpolymers as evidenced by electron microscopy.121 Nevertheless,a cross-comparison of Triton X-100 versus centrifugation orincreasing protein concentration showed similar results for 37,42, and 20.97 Furthermore, Triton X-100 has been used in con-trol 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 anaggregator will result in a loss of activity at any drug con-centration.In vitro measurements of GTP hydrolysis by FtsZ are

complicated by the complexity of the biological couplingbetween GTPase activity and FtsZ protofilament formation.A thorough reexamination of several reported FtsZ inhibitorsindicates two are aggregators (e.g., 7 and 37); one is a PAINscompound (e.g., 32), and several inhibitors have activity thatvary significantly from earlier reports (e.g., 9).97 Aggregation isa problem for in vitro assays, such as GTPase activitymeasurements and light scattering experiments, in which anonspecific aggregation effect of small molecule inhibitors withFtsZ monomers impedes polymerization. FtsZ protofilamentsand bundles can be observed by electron microscopy; howeverthis technique is unable to decouple whether a decreasing FtsZpolymerization rate and the change in the number of proto-filaments are a result of aggregation or an inhibitor binding toFtsZ specifically. FRET assays can be used to transcend thislimitation. One population of FtsZ proteins can be labeled witha donor fluorophore (a FRET donor) and another with anacceptor fluorophore (a FRET acceptor). Polymerization ofFtsZ monomers creates a spatial distribution of fluorophores; adonor and acceptor positioned adjacent to each other willproduce a FRET signal.207 An inhibitor that disrupts FtsZpolymerization may reduce the FRET signal.Cell filamentation arises in response to alterations in the

topological state or structure of DNA. As mentioned in section3.2.3, when the SOS response is triggered in response to DNAdamage, SulA inhibits FtsZ polymerization (Table 2) andcell filamentation occurs. However, bacteria can filament viaSulA-independent mechanisms after the SOS response istriggered by DNA damage, perturbation of DNA topology,and stalling of the DNA replication fork.185,208 The first step inthe SOS response is controlled by the RecA-activated self-cleavage of LexA, a transcriptional repressor of all the SOSresponse genes.208 Therefore, the use of a ΔsulA mutant with anoncleavable LexA repressor (lexA (ind−)) is more informativethan a ΔsulA mutant, as the activation of all of the SOSresponse genes and their contribution to SulA-independentfilamentation 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 evenlyspaced along the length of the filament),209 suggesting thatinhibiting Fts proteins (other than FtsZ) produces a filamentousphenotype. Inhibiting some clinically relevant antibacterialtargets (such as inhibitors of peptidoglycan biosynthesis andDNA supercoiling) produces a filamentous cell phenotype.



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Consequently, validating that FtsZ inhibitors do not targetpenicillin-binding proteins or DNA gyrase is an important stepin characterizing new drugs.210,211 Surprisingly, some FtsZinhibitors, such as the zantrins, compounds 37−41, andhemi-10, do not filament cells (or do only very minimally),yet they mislocalize the Z-ring.122 These observations lead toquestions surrounding whether FtsZ is the primary target ofthese compounds.Additionally, the mislocalization of the Z-ring can be

attributed to many factors that precede its formation. Positiveprotein regulators of FtsZ are involved in localizing FtsZ to thecenter of the cell (Table 2). Consequently, their inhibitioncould result in a misplacement of the Z-ring. As discussedpreviously in section 3.3, the localization of cytoskeletal and celldivision proteins has been shown to be sensitive to membranedepolarization. The mislocalization of the Z-ring can alsobe attributed to compromised membranes and a disruptedmembrane potential.171 Therefore, the characterization of apossible FtsZ inhibitor should be accompanied by determiningwhether it affects the membrane.4.6.2. Strategies To Improve Screening and Character-

ization Approaches for New FtsZ Inhibitors. To improve thechances of identifying new FtsZ inhibitors, new screeningmethods should explore new genetic approaches and in vivotechniques. In vivo high-throughput screens with clever cell-based reporter systems provide information about antibacterialpotency. Using overexpression strains of FtsZ or an enzymethat interacts with it (Table 2) would distinguish smallmolecule inhibitors whose antibacterial activity is reversed byoverexpression of these enzymes. In accordance with protein−protein interactions, a small molecule activator of SulA (“fake”DNA damage), ClpXP (proteolytic activity), or SlmA (pseudochromosome mis-segregation) would theoretically cause anindirect stalling of the Z-ring.Many recent detection techniques utilizing new instruments

could be implemented for in vivo screens. Microscopes andflow cytometers have been used for high-throughput screeningby incorporating stage attachments and detectors that facilitatethe use of 96-well plates. Using a 96-well plate microscopyassay, one could observe the cell filamentation and mislocaliza-tion of the Z-ring on a large scale that employs more individualcompound treatments than are possibly using serial, one-at-a-time assays. Flow cytometers can simultaneously measure manyaspects of cell physiology (i.e., the length of cells) and theintensity of a chosen fluorescent probe (i.e., 60) in seconds.An experiment could entail grouping all of the in vivo char-acteristics of a small molecule into one flow cytometryexperiment using a ΔsulA lexA (ind−) mutant to control forSOS-dependent cell filamentation, a fluorescently tagged FtsZto observe Z-ring mislocalization, and a quantifiable DNA stain(such as Picogreen) to quantify the number of chromosomesper nucleoid. The translation of biophysical assays from otherareas of biology could be useful for screening small moleculelibraries to identify new inhibitors of FtsZ.

5. SUMMARY AND FUTURE DIRECTIONSBacteria use a variety of regulatory mechanisms to influence theinitiation and progression of cell division, many of whichultimately hinge on the essential cell division protein FtsZ.These mechanisms ensure spatiotemporal coordination of celldivision and other biological processes necessary for cellviability. Recent studies have uncovered new biochemistryrelated to cell division that can be exploited for antibiotic

development; however many aspects of this regulatory networkremain enigmatic. Its central position in cell division highlightsFtsZ as a prime candidate for chemotherapeutic strategies, andyet after nearly 2 decades of research on FtsZ inhibitors noinhibitors have emerged for clinical use.The plethora of papers on FtsZ antagonists and lack of

multiple, potent inhibitors of FtsZ suggest that finding smallmolecules to target this protein is challenging. When theactivity of these molecules is considered in the context of themany potent inhibitors of tubulin that have been clinicallydeveloped, the medicinal chemistry of FtsZ appears to still bestuck in very early stages of development and the field is facedwith an important question: Why does this large discoverybandgap exist?One possible explanation is that FtsZ is a much harder

protein to drug than tubulin. Comparisons of the crystal struc-tures indicate that tubulin contains numerous regions forbinding small molecules, while FtsZ has fewer regions in whichmolecules can bind, thereby reducing the probability of findingan inhibitor that binds FtsZ antagonistically. Another relatedexplanation is that there is still very little crystal structure dataon FtsZ compared to tubulin. Although there are >30 crystalstructures deposited in PDB, ∼30% of them are of S. aureusFtsZ and only one has a non-nucleotide small molecule boundin the structure, 42, which provides limited information on howcompounds can alter FtsZ structure and activity. 42 binds toS. aureus FtsZ in a flat orientation and appears to affectpolymerization by shifting the H7 helix marginally. However,the general lack of crystallographic data makes it difficult tolearn how to design better inhibitors using structural biologydata. Additional structural biology data of FtsZ bound to themost potent inhibitors could provide insight into designprinciples; however the lack of available potent FtsZ inhibitorslimits this approach.A second possibility centers upon current chemical libraries,

which do not often include new molecular entities that containstructural features that are a hallmark of successful clinicalantibiotics. Commercial molecular libraries remain locked in amindset of Lipinski’s rules, which has been successful foridentifying inhibitors of individual proteins and compoundsactive in eukaryotic cells (e.g., mammalian cells) but is notparticularly effective for targeting bacteria. Other sets of rulesfor molecular properties that improve bacterial uptake (e.g.,Moser’s rules)212 may be a more helpful tool for curating smallmolecule libraries for families of molecules that may haveantibacterial properties. Specifically, these molecules may haveimproved transport properties across bacterial membranes andmay be poor substrates for transport out of cells through effluxpumps. Limitations of chemical space in synthetic chemicallibraries would benefit from following other druglikeness rulesfor antibiotic drug development.One way to fill this void in the chemical space of commercial

libraries is to refocus on natural products and libraries thatcontain secondary metabolites; here dereplication and meta-genomic techniques may be particularly effective. Onechallenge with FtsZ however remains that it is widely conservedamong bacteria, which reduces the possibility that bacteriaevolved secondary metabolites to inhibit FtsZ in other cells. Forthis mechanism to be possible, antibiotic-producing bacteriawould require a chaperone that keeps the inhibitor bound untilit is secreted. Alternatively, bacteria may evolve mutated formsof FtsZ that have reduced binding to secondary metabolitesthat they secrete to inhibit the growth of competing bacteria.



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However, the lack of divergence among FtsZ indicates that itmay be difficult to mutate the protein and retain its enzymaticfunction, assembly into filaments, force generation, andcontacts with other proteins. These characteristics may haveprevented genetic drift of FtsZ and dampened the developmentof mechanisms of chemical warfare for inhibiting cell divisionthrough targeting FtsZ. These considerations reduce thefeasibility of secondary metabolites that evolved to targetFtsZ. Perhaps some of the exciting new techniques forsecondary metabolite identification213 will turn up newcompounds with activity against FtsZ and motivate the fieldto 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 studiesto confirm on-target binding and rule out indirect mechanismsof FtsZ antagonism (e.g., triggering the SOS pathway,aggregation and promiscuous binding to proteins, and alteringcell membrane potential). Cell filamentation and Z-ringmislocalization are characteristic phenotypes of FtsZ inhibitors;however they can be caused by many different mechanisms.FtsZ studies reveal an aspect of medicinal chemistry that isimportant for drug design and development: the role ofstructural biology in confirming and characterizing bindinginteractions to the target. The lack of small molecules that havebeen solved in cocrystal structures to FtsZ indicates either avery limited repertoire of current drugs that are bona fideinhibitors of FtsZ or an intrinsic challenge of crystallizing FtsZand 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 areconnected to FtsZ and cytokinesis. There are a variety of invivo assays that can be used (or new methods that can beengineered) to qualitatively or quantitatively measure theinhibition of cell division and are compatible with high-throughput screening methods. Bacterial cell division remainsan active area of fundamental research, and inhibitors of specificproteins that participate in this process may be important toolsfor studying the biochemical and biophysical mechanisms thatare involved. Although 42 has become the canonical FtsZinhibitor, it is only effective against S. aureus FtsZ and, for thereasons highlighted earlier, it has provided limited under-standing of how to design molecules to target this protein.New families of clinical antibiotics against Gram-negativebacteria that display multidrug resistance214,215 may motivatethe discovery of cell division inhibitors against these organisms.FtsZ remains an attractive target for inhibiting division inGram-negative bacteria. Potent antagonists may have a dual usein understanding how bacteria coordinate the multiple steps ofcell division and as antimicrobial agents, which may lead to newclinical antibiotics for chemotherapies.

■ 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.

Author Contributions#K.A.H. and T.M.A.S. contributed equally to this article.

NotesThe authors declare no competing financial interest.

Biographies

Katherine A. Hurley received her B.S. in Chemistry with apharmaceutical emphasis at University of CaliforniaDavis, CA, andcompleted research studies under the supervision of Professor Jared T.Shaw. She is currently pursuing a Ph.D. degree in PharmaceuticalSciences from the School of Pharmacy at the University ofWisconsinMadison, WI, under the supervision of Douglas Weibel.Her present research involves discovering and characterizing newantibiotics as chemotherapeutic agents and chemical biology probes.

Thiago M. A. Santos obtained his B.S. in Biological Sciences and aM.S. degree in Agricultural Microbiology from Universidade Federalde Vicosa, Brazil, and pursued predoctoral studies in the College ofVeterinary Medicine at Cornell University, NY. He is currentlypursuing a Ph.D. degree in the Microbiology Doctoral TrainingProgram at the University of WisconsinMadison, WI, under thesupervision of Douglas Weibel. His current research deciphers themolecular mechanisms of protein localization in bacteria and the modeof action of novel antimicrobial drugs.

Gabriella M. Nepomuceno received her B.S. in Chemistry atUniversity of CaliforniaSanta Cruz, CA, and completed researchstudies under the supervision of Professor Bakthan Singaram. Shereceived a Ph.D. degree in Chemistry from the University ofCaliforniaDavis, CA, under the supervision of Jared T. Shaw. Herresearch involved designing and synthesizing molecular probes inchemical biology and organic methodology.

Valerie Huynh received her B.S. in Pharmaceutical Chemistry fromthe University of CaliforniaDavis, CA, under the supervision ofJared T. Shaw. She received a M.S. degree in PharmaceuticalChemistry at the same institution. Her research involved synthesizingmolecular probes for medicinal chemistry. Currently, she is a ResearchAssociate at Gilead Sciences, Inc.

Jared T. Shaw received his Ph.D. in Chemistry from Keith Woerpel atUniversity of CaliforniaIrvine and then moved to HarvardUniversity, MA, as an NIH Postdoctoral Fellow with David Evans.He became an Institute Fellow at the Institute for Chemistry and CellBiology (ICCB) at Harvard Medical School where he helped foundthe Center for Chemical Methodology and Library Development(CMLD), which later became part of the Broad Institute of Harvardand Massachusetts Institute of Technology. He is currently anAssociate Professor of Chemistry at the University of CaliforniaDavis, CA, and he currently works on the development of newmethods for the synthesis of natural products and other complexmolecules that modulate biological phenomena.

Douglas B. Weibel received his B.S. degree in Chemistry from theUniversity of Utah, was a Fulbright Fellow at Tohoku University,Japan (with Yoshinori Yamamoto), and received his Ph.D. inChemistry from Cornell University, NY (with Jerrold Meinwald).He was a Postdoctoral Fellow at Harvard University, MA (withGeorge Whitesides). He is currently an Associate Professor ofBiochemistry, Chemistry, and Biomedical Engineering at theUniversity of WisconsinMadison, WI, and his research spans thefields of biochemistry, biophysics, chemistry, materials science andengineering, and microbiology.

■ ACKNOWLEDGMENTS

Due to space constraints, we were unable to cite all of theresearch on FtsZ; any occlusions were unintentional. Research onantibiotics in the Weibel laboratory has been supported by theHuman Frontiers Science Program (Grant RGY0076/2013), theNIH (Grant 1DP2OD008735), the Wisconsin Alumni Research



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mailto:[email protected]

mailto:[email protected]


Foundation, and the USDA (Grant WIS01594). Research oninhibitors of cell division in the Shaw laboratory is supported byNIH/NIAID (Grants R01A108093, R01A08093-04S1).

■ ABBREVIATIONS USEDANS, 8-anilinonaphthalene-1-sulfonic acid; ADME, absorption,distribution, metabolism, and excretion; CCCP, carbonylcyanide m-chlorophenyl hydrazone; DAPI, 4′,6-diamidino-2-phenylindole; EGS, external guide sequence; FDA, Food andDrug Administration; FRAP, fluorescence recovery afterphotobleaching; FRET, fluorescence resonance energy transfer;GDP, guanosine diphosphate; GTP, guanosine triphosphate;pN, piconewton; OTBA, 3-{5-[4-oxo-2-thioxo-3-(3-trifluoromethylphenyl)thiazolidin-5-ylidenemethyl]furan-2-yl}-benzoic acid; PAIN, pan-assay interference; ROCS, rapidoverlay of chemical structures; SAR, structure−activity relation-ship; UV, ultraviolet

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