Targeting the Bacterial Division Protein FtsZ

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1 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, 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 localization 10 (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. The essential cytoskeletal cell division protein FtsZ (named after the filamenting temperature-sensitive mutant Z) is an essential GTPase structurally related to eukaryotic tubulins 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 biology 16,19,20 and inhibition with small molecules have been discussed Specifically, targeting FtsZ with small molecules as a defense against tuberculosis has also been extensively reviewed 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, American Chemical Society 6975

2 Journal of Medicinal Chemistry 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 ). 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 6976

3 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 H5 29 )(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 anddisassembly,suchasminc, 33 FtsA, 34,35 ZipA, 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 6977 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 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

4 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 remodeling (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 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 essential 48,57,58 (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 separate 49,50,57,59 (Figure 2B). The divisome machinery is essential and appears to be widely conserved among bacteria 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-cell 60,61 and (2) nucleoid occlusion proteins prevent division from occurring over segregating chromosomes 62,63 (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 6978 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-cell 65 (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-ring 66 (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 FtsZ Biophysics and Mechanics. An average E. coli cell during log-phase growth contains 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 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 pnis 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

5 Table 2. Proteins That Regulate the Formation of the Z-Ring in Bacteria a (1) Positive Regulators of Z-Ring Formation 51,69 FtsA b -Membrane anchor supports assembly and stabilization of the Z-ring. It is also important for the recruitment of downstream proteins necessary for divisome maturation. 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 inhibit formation of the Z-ring in a ClpP-independent fashion by physically blocking the assembly of FtsZ filaments 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 its turnover. 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 between FtsZ 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-ring formation 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 that cells 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 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. b It 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 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 ,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 activity and the initial constriction of tubular liposomes in the presence of this GTPase-dead mutant FtsZ 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 6979 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

6 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 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 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 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 toinhibiting Z-ringformation and suppressing the expression of FtsZ mrna. 92, 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 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 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 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

7 Journal of Medicinal Chemistry Table 3. Summary of Reported FtsZ Inhibitors Discussed in the Texta 6981

8 Table 3. continued 6982

9 Table 3. continued 6983

10 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 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 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 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 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 K i 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 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 6984

11 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 cells 118 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 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 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 PC (42) 124 from the starting inhibitor 3-methoxybenzamide (43) was the first non-nucleotide inhibitor of FtsZ to be cocrystallized with FtsZ was initially described as stabilizing FtsZ protofilaments 127 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, 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 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 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) 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 and ,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 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 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-1- naphthalenesulfonate (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 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 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[2,3- a]quinolizin-7-one inhibitors (compounds 1 (64)and10b(65))

12 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 ) of ZipA 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 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 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 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 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 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 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 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 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 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. 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 6986