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Perspective pubs.acs.org/jmc Targeting the Bacterial Division Protein FtsZ Katherine A. Hurley,†,# Thiago M. A. Santos,‡,# Gabriella M. Nepomuceno,§ Valerie Huynh,§ Jared T. Shaw,*,§ and Douglas B. Weibel*,‡,∥,⊥ † Department of Pharmaceutical Sciences, University of 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 mechanical properties of bacterial cells. The essential protein FtsZ is a central player in the cytoskeletal family, forms a cytokinetic ring at mid-cell, and recruits the division machinery to orchestrate cell division. Cells depleted of or lacking functional FtsZ do not divide and grow into long filaments that eventually lyse. FtsZ has been studied extensively as a target for antibacterial development. In this Perspective, we review the structural and biochemical properties of FtsZ, its role in cell biochemistry and physiology, the different mechanisms of inhibiting FtsZ, small molecule antagonists (including some misconceptions about mechanisms of action), and their discovery strategies. This collective information will inform chemists on different aspects of FtsZ that can be (and have been) used to develop successful strategies for devising new families of cell division inhibitors. 1. INTRODUCTION: TARGETING THE BACTERIAL PROTEIN FtsZ An increase of multidrug resistance to antibiotics among pathogenic strains of bacteria and the lack of innovation in the discovery of new antibacterial agents punctuate the need for new chemotherapeutic strategies. One approach to new strategies is the identification, characterization, and exploration of new molecular targets for antibiotic development, which is currently in vogue. Historically, all known clinical antibiotics target one of the following bacterial structures and cellular processes: (1) DNA replication; (2) transcription; (3) translation; (4) peptidoglycan biosynthesis; (5) folate biosynthesis; (6) the cytoplasmic membrane.1,2 An important, unanswered question is whether additional classes of mechanisms and targets exist for developing new families of antibiotics. The bacterial cytoskeleton is one such family of targets for which clinical antibiotics have not yet emerged. The cytoskeleton is an ancient cellular invention that probably precedes the divergence between eukaryotes and prokaryotes.3 The bacterial cytoskeleton consists of families of proteins essential for the physiological and structural properties of cells, including cell division,4,5 cell wall growth,6,7 cell shape determination/ maintenance,8,9 DNA segregation,10 and protein localization10 (Table 1). Because its integrity is important to cell viability, the bacterial cytoskeleton has been a topic of discussion for the development of antibacterial compounds over the past 2 decades. © 2016 American Chemical Society The essential cytoskeletal cell division protein FtsZ (named after the filamenting temperature-sensitive mutant Z) is an essential GTPase structurally related to eukaryotic tubulins11−13 and highly conserved in bacteria and archaea.14,15 During cell division, FtsZ forms a ringlike structure at the site of division and functions as a scaffold for the assembly of a multiprotein complex (referred to as the “divisome”) essential for cell viability. Not surprisingly, FtsZ, as well as proteins that interact directly with and regulate the activity of FtsZ, has emerged as a prime target for antibacterial development.16 The use of FtsZ as an antibacterial drug target has been reviewed,17,18 and its structural biology16,19,20 and inhibition with small molecules have been discussed.21−25 Specifically, targeting FtsZ with small molecules as a defense against tuberculosis has also been extensively reviewed.26−28 In this review, we explore the latest developments of classes of small molecules and inhibitors targeting FtsZ and evaluate the challenges and future directions of this field of antibiotic research. 2. STRUCTURE AND FUNCTION OF FtsZ 2.1. FtsZ Structure. FtsZ shares 40−50% sequence identity across most bacterial and archaeal species and has a threedimensional structure that is similar to the structure of α- and Received: July 14, 2015 Published: January 12, 2016 6975 DOI: 10.1021/acs.jmedchem.5b01098 J. Med. Chem. 2016, 59, 6975−6998 Journal of Medicinal Chemistry Perspective Table 1. Examples of Key Components of the Bacterial Cytoskeletona cytoskeletal proteinb Tubulin-like FtsZ TubZ Actin-like FtsA MreB ParM Intermediate Filaments crescentin Walker A “Cytoskeletal” ATPases MinD ParA function/remarks -Is the structural subunit of the Z-ring -Recruits downstream proteins involved in cell division and cell wall synthesis and remodeling -Involved in DNA segregation -Membrane tether required for Z-ring assembly -Recruits downstream proteins involved in cell division and cell wall synthesis and remodeling -Destabilizes FtsZ filaments on the membrane, enabling rapid reorganization of the filament network222 -Required for cell shape determination (morphogenesis) and maintenance -Is also implicated in chromosome segregation and cell polarity -Participates in DNA segregation -Responsible for the asymmetric cell shape in some bacteria (e.g., it is an essential determinant of the curved shapes of C. crescentus cells) -Involved in positioning the Z-ring at mid-cell -Participates in DNA segregation a This is not a comprehensive list. The bacterial cytoskeleton consists of other families of proteins or protein homologues that are absent from this list. Further information can be found reviewed in ref 223. bSome of these cytoskeletal proteins are essential and widespread among bacteria. However, some of them are exclusive to specific bacteria groups. See the text and refer to the cited literature for an additional explanation. The referencing is not exhaustive for the best-studied proteins included in the list. Figure 1. FtsZ is the ancestral homologue of tubulin and is highly conserved in bacteria. Top: A representation of the monomers of FtsZ and β-tubulin with GDP (in orange) bound in the active site. Left to right: S. aureus (PBD code 3VOA),85 M. jannaschii (PBD code 1FSZ),29 and S. scrofa (PBD code 1TUB).30 The direction of the polymerization is indicated based on the axis of the protofilament. Bottom left: A representation of the dimerization of two monomers of FtsZ from S. aureus (PBD code 3VOA)85 and M. jannaschii (PBD code 1W5A)29 with GDP (in red) bound in the active site. Each monomer is represented as a different shade of green to facilitate visualization, and GDP is represented as electrostatic spheres in brick red. Bottom right: A demonstration of the dimerization of one monomer of β-tubulin (dark green) with GDP bound in the active site and one monomer of α-tubulin (light green) with GTP bound in the active site from S. scrofa (PBD code 1TUB).30 The direction of the polymerization is indicated based on the axis of the protofilament. These representations were generated using PyMOL (version 1.5.0.4). β-tubulin3,13,29,30 (Figure 1). Despite structural and functional similarities, FtsZ is a distant ancestral homolog of tubulin with an amino acid sequence that is <20% identical.3,13,31,32 Crystallographic analysis of FtsZ from the hyperthermophilic methanogen Methanocaldococcus jannaschii (formerly Methanococcus jannaschii) revealed the presence of two domains 6976 DOI: 10.1021/acs.jmedchem.5b01098 J. Med. Chem. 2016, 59, 6975−6998 Journal of Medicinal Chemistry Perspective Figure 2. Spatiotemporal regulation of the Z-ring in different groups of bacteria. (A) Diagram summarizing the hierarchical recruitment of cell division proteins in E. coli. FtsZ and the early cell division proteins localize to the division site before cell septation starts. The proteins are recruited to the Z-ring in a sequential and approximately linear pathway. The requirement of an upstream protein for localization of a downstream protein to the Z-ring was deduced from various studies of genetics, biochemistry, and microscopy. Proteins that regulate the assembly of the Z-ring are shown in green (positive regulators) and red (negative regulators). Peptidoglycan-specific amidases AmiA, AmiB, and AmiC play an important role in cleaving the septum to release daughter cells after division in E. coli. AmiB and AmiC localize to the division site, whereas AmiA (not included in the diagram) is diffusely localized in the periplasm.216 This diagram was redrawn from refs 48, 51, and 217. (B) A cartoon illustrating some of the bacterial mechanisms for positioning of the Z-ring during cell division. In E. coli, Min proteins (MinCDE) oscillate between the cell poles, creating an inhibition zone (green shaded area) and preventing Z-ring (in red) polymerization near those poles.60,61,218 (For simplicity, the dynamic behavior of the Min system is omitted.) In addition, nucleoid occlusion, mediated by the protein SlmA, creates an inhibition zone (blue shaded area) along the cylindrical region of the cell and prevents Z-ring assembly over the nucleoid.63 Inset: A cartoon depicting the predicted organization of the E. coli divisome. Cell division is initiated with the polymerization of FtsZ into the Z-ring onto which the divisome apparatus assembles. The cartoon of the divisome was adapted from refs 48 and 217. Similar to E. coli, the B. subtilis Min system (composed of MinCDJ and DivIVA) creates a zone of inhibition (purple shaded area) that prevents Z-ring assembly at the cell poles. However, in B. subtilis, MinCDJ localizes to the cell poles in a DivIVAdependent manner and does not undergo the characteristic dynamic oscillatory behavior observed in E. coli.219,220 In addition to the Min system, the protein Noc mediates nucleoid occlusion (blue shaded area), preventing divisome assembly from occurring over segregating chromosomes.62 In C. crescentus, the protein MipZ (yellow shaded area) coordinates chromosome segregation and cell division in response to both spatial and temporal cues. The assembly of Z-ring is coincident with the subcellular position that exhibits the lowest concentration of MipZ. Prior to chromosome replication, MipZ and FtsZ are localized to the opposite poles of the cell. MipZ forms a complex with proteins involved in chromosome partitioning. Following duplication of the chromosomal replication origin region (oriC), MipZ migrates toward the opposite cell pole creating a bipolar gradient displacing the FtsZ at the poles and directing formation of the Z-ring toward mid-cell.65,221 In S. pneumoniae, the protein MapZ forms a ringlike structure (in orange) positioned at mid-cell (and at future division sites), marking the cell division site and positioning FtsZ.66 See the text for an additional explanation. connected by a long central helix (H7)13 (also designated as helix H529) (Figure 1). The amino-terminal portion of the protein consists of a six-stranded β-sheet sandwiched by two helices on one side and three on the other and contains the GTPase domain. This domain is conserved between FtsZ and tubulin; however, it is different in other classic GTPases.13 The carboxy-terminal domain consists of a four-stranded β-sheet in contact with helix H7 and supported by two helices on one side while the other side is exposed to the solvent.29 The conserved C-terminal tail of FtsZ mediates specific interactions between FtsZ and auxiliary proteins that regulate divisome assembly and disassembly, such as MinC,33 FtsA,34,35 ZipA,34−36 EzrA,37 ClpX,38 SepF,39 and FtsZ itself.40 FtsZ assembles into protofilaments that form tubules, sheets, and minirings in vitro.41,42 Super-resolution microscopy studies in vivo demonstrate that the Escherichia coli Z-ring adopts a compressed helical conformation with variable helical length and thickness of ∼110 nm.43 Electron cryotomographic reconstructions of dividing Caulobacter crescentus cells revealed that the Z-ring consists of short protofilaments that are ∼100 nm in length and randomly spaced near the division site and positioned ∼16 nm away from the inner membrane.44 More recent electron cryomicroscopic and cryotomographic studies in E. coli, C. crescentus, and constricting liposomes confirmed the distance of the protofilaments positioned from the inner membrane and revealed that in both bacterial species the Z-ring is probably a continuous structure consisted of single-layered bundles of FtsZ that are 5−10 filaments wide.45 2.2. Role of FtsZ in Cell Division. Bacterial cell division is a complex process that requires accurate identification of the division site, positioning of the division machinery, and 6977 DOI: 10.1021/acs.jmedchem.5b01098 J. Med. Chem. 2016, 59, 6975−6998 Journal of Medicinal Chemistry Perspective B. subtilis. However, many bacteria lack both canonical systems for positioning FtsZ. For example, in the Gram-negative bacterium C. crescentus, spatiotemporal assembly and placement of the Z-ring require MipZ.65 MipZ is an ATPase that associates with the origin region of chromosomes and (in a manner analogous to the Min system) directly guides FtsZ positioning and polymerization into the Z-ring at mid-cell65 (Figure 2B). In the Gram-positive pathogen Streptococcus pneumoniae, MapZ localizes at the division site prior to FtsZ, interacts directly with FtsZ, and guides positioning of the Z-ring66 (Figure 2B). MapZ is a single-passage transmembrane protein that is conserved among Streptococcaceae and other Lactobacillales and forms ringlike structures positioned at mid-cell and at future division sites. In addition to its dual role in marking the cell division site and positioning FtsZ, the balance between the phosphorylated and dephosphorylated forms of MapZ may be important for controlling Z-ring stability and regulation of constriction.66 In addition to these specific protein-based systems that control FtsZ dynamics, other positive and negative regulators interact with FtsZ to modulate structure and function of the divisome in response to the nutritional and developmental state of the cell (Table 2). These regulatory systems for positioning of FtsZ have adapted to different environments, cell shapes, and developmental behaviors and emphasize the importance of coordinating the correct timing of cell division and chromosome segregation. Many studies have indicated that unknown regulators of bacterial FtsZ may be awaiting discovery. 2.5. FtsZ Biophysics and Mechanics. An average E. coli cell during log-phase growth contains ∼15 000 molecules of FtsZ.67 The Z-rings assemble at the future site of division in E. coli daughter cells before the Z-ring is fully constricted in the parental cell.59 This observation suggests that future division sites in daughter cells become competent for assembly of the divisome prior to the complete division of the mother cell. In addition, FRAP experiments in E. coli cells demonstrate that the Z-ring is a dynamic structure, continuously remodeled by exchanging subunits with the cytoplasmic pool of FtsZ (halftime of recovery ∼30 s). The kinetics of FtsZ turnover in vivo is tightly coupled to GTP hydrolysis, as mutant cells with reduced GTPase activity of FtsZ show ∼9-fold slower turnover of FtsZ into protofilaments.68 In vitro studies performed at physiological conditions show that purified E. coli FtsZ assembles into protofilaments and hydrolyze GTP at a rate of ∼5 molecules per min per FtsZ, demonstrating that the GTPase activity of FtsZ in vitro is very slow.67 In addition to functioning as an essential molecular scaffold for recruitment and organization of the other cell division proteins to the division site,48,69 FtsZ generates a contractile force that constricts tubular liposomes in vitro.70−72 FtsZ is thought to act as an important source of the constriction force required for cytokinesis during cell division; however, the mechanism by which FtsZ generates mechanical force and promotes invagination of the cell wall during division remains unclear. Previous studies with purified FtsZ have shown that the GTP-bound FtsZ assembles into straight or gently curved filaments, while the GDP-bound FtsZ forms highly curved filaments,41,42 suggesting that the difference in the intrinsic curvature of FtsZ filaments provides a mechanism for generating mechanical force for cell division. Models describing the process of cell growth and Z-ring contraction in E. coli cells predict that a force of ∼8 pN is sufficient to pull the cell wall inward at the division site to initiate the constriction, and forces of 8−80 pN could lead to cell coordinated constriction of the inner membrane and the cell wall (i.e., cytokinesis). With few exceptions,46,47 this essential process is initiated with the polymerization of FtsZ into a filamentous, ringlike structure (referred to as the Z-ring) that is located in the cytoplasm peripheral to the membrane and close to the division site.5,43−45 Concomitant with and following its polymerization, the Z-ring recruits and coordinates a series of auxiliary proteins that perform diverse roles in cell division and cell wall biosynthesis and remodeling48−51 (Figure 2A). Depletion of FtsZ in rod-shaped bacteria, such as the Gramnegative E. coli, produces long, filamentous cells due to the continued growth of cells that are no longer dividing.4 Cocci-shaped bacteria, such as the Gram-positive pathogen Staphylococcus aureus, increase in volume up to 8-fold when depleted of FtsZ.52 In both cases, cells are unable to divide; continued growth makes them enlarged and sensitive to changes in the physical properties of their environment, and the cells eventually lyse. Drugs that affect the positioning, activity, and interaction of FtsZ with other division proteins cause cell lysis and may be useful as antibiotics. 2.3. FtsZ Dynamics during the Division Cycle. The spatiotemporal regulation of Z-ring formation requires a complex and concerted network of proteins that modulate assembly and activity of FtsZ to ensure that the division process is tightly coordinated with DNA replication, chromosome segregation, and cell elongation.48,53−56 The molecular details underlying the synchronicity of these processes are not completely understood; however, structural and cell biology research over the past 2 decades has elucidated important structural, functional, and regulatory aspects of these mechanisms and how they are coordinated. The division process in E. coli cells requires at least 14 major cytoplasmic, membrane, and periplasmic proteins, of which ∼10 are essential48,57,58 (Figure 2A). FtsZ and the other cell division proteins, as well as their regulators, are recruited to mid-cell in a hierarchical order to form the functional divisome, a ringlike multiprotein complex that constricts during the process of division and disappears when the cells separate49,50,57,59 (Figure 2B). The divisome machinery is essential and appears to be widely conserved among bacteria. 2.4. Regulatory Proteins That Position FtsZ in Bacteria. Rod-shape bacteria (such as E. coli and the Grampositive bacterium Bacillus subtilis) use at least two coordinated biochemical systems to accurately position the Z-ring at the mid-cell and ensure that divisome formation is timed to occur at the final stage of the cell cycle: (1) the Min system of proteins prevents aberrant division at regions other than the mid-cell60,61 and (2) nucleoid occlusion proteins prevent division from occurring over segregating chromosomes62,63 (Figure 2B). Importantly, in the absence of these two negative regulators of Z-ring positioning, both E. coli and B. subtilis still have a bias for Z-ring formation at mid-cell,62,63 suggesting that additional mechanisms may influence FtsZ assembly at the mid-cell and coordinate chromosome segregation and cell division. A recent study provided evidence of an additional positional marker in E. coli cells grown in minimal media and lacking functional Min and nucleoid occlusion systems.64 In particular, the authors identified that the Ter macrodomain region of the chromosome acts as a landmark for the Z-ring in the presence of the chromosomal terminus organization protein MatP and the cell division proteins ZapA and ZapB.64 Much of the mechanistic insight on the spatiotemporal regulation of the Z-ring has come from studies of E. coli and 6978 DOI: 10.1021/acs.jmedchem.5b01098 J. Med. Chem. 2016, 59, 6975−6998 Journal of Medicinal Chemistry Perspective Table 2. Proteins That Regulate the Formation of the Z-Ring in Bacteriaa (1) Positive Regulators of Z-Ring Formation51,69 b FtsA SepF ZapA,B,C,D ZipA ClpX/ClpXP CrgA EzrA GdhZ KidO OpgH MciZ MinC MipZ MapZ Noc SlmA SulA UgtP YneA -Membrane anchor supports assembly and stabilization of the Z-ring. It is also important for the recruitment of downstream proteins necessary for divisome maturation. -Required for proper morphology of the divisional septum. It has overlapping roles with FtsA in Z-ring assembly. -Mediates additional stabilization of the Z-ring. -Secondary membrane anchor that together with FtsA supports assembly of the Z-ring. (2) Negative Regulators of Z-Ring Formation -Helps modulate the equilibrium between the cytoplasmic pool of unassembled FtsZ and polymeric FtsZ through degradation.38 ClpX chaperone can also inhibit formation of the Z-ring in a ClpP-independent fashion by physically blocking the assembly of FtsZ filaments.153−155 -Important for coordinating cell growth and division. It regulates the dynamics of Z-ring formation and affects both the timing of FtsZ expression and its turnover.224 -Modulates the position of the Z-ring during cell division and plays a role in coordinating cell growth and division.69 -NAD-dependent glutamate dehydrogenase that controls Z-ring disassembly by stimulating the GTPase activity of FtsZ.225 -Coordinates cellular or developmental activities with the availability of NADH. KidO bound to NADH is thought to destabilize lateral interactions between FtsZ protofilaments. It has been recently proposed to work in synergy with GdhZ to trigger Z-ring disassembly.225,226 -Glucosyltransferase that functions as a nutrient-dependent antagonist of the Z-ring. OpgH is thought to sequester FtsZ from growing polymers. Blocks Z-ring formation to coordinate cell growth and cell division.227 -Inhibits Z-ring formation by capping the minus end of FtsZ filaments and shortening the filaments.228 -Important for positioning the Z-ring at mid-cell.60,61,218 -Required for positioning the Z-ring at mid-cell.65 -Important for Z-ring formation and positioning at mid-cell. It is also involved in the regulation of cytokinesis.66 -Inhibits Z-ring formation over segregating chromosomes.62 -Analogous to Noc, it inhibits Z-ring formation over segregating chromosomes.63 -Negative modulator of Z-ring expressed in response to DNA damage as part of the SOS system.165,168,169 -Similar to OpgH, this glucosyltransferase inhibits cell division by blocking Z-ring formation in a growth rate-dependent fashion. This cellular sensor ensures that cells reach the appropriate mass and complete chromosome segregation prior to cytokinesis.229 -Analogous to SulA, it regulates cell division through the suppression of Z-ring formation during the SOS response.230 a Some of these proteins and the molecular systems that they compose are widespread among bacteria. However, some of them are exclusive to specific groups of bacteria. See the text and refer to the cited literature for an additional explanation. The referencing is not exhaustive for the beststudied proteins listed. bIt was recently demonstrated that FtsA has a dual, antagonistic role on the FtsZ filament network. FtsA is involved in recruitment of FtsZ filaments to the membrane, but it also provides a negative regulation by causing fragmentation of FtsZ polymers, allowing the rapid disassembly of FtsZ filaments.222 division by creating a reasonably accurate septal morphology.73 Molecular simulations of FtsZ dynamics estimate that FtsZ can generate a value of ∼20−30 pN per polymerized monomer when GTP is hydrolyzed,74 which is sufficient to direct cell− wall invagination during division and cause membrane vesicle formation from liposomes in vitro.70−72,75 Although mathematical models quantitatively support the “hydrolyze-and-bend” mechanism for force generation of the Z-ring,41,76,77 slightly curved GTP-FtsZ filaments (without GTP-hydrolysis) are capable of supplying a force of ∼10 pN, suggesting that nucleotide hydrolysis might not be required for membrane bending by FtsZ.74,75 This observation could explain the occasional division events observed in cells containing a mutant version of FtsZ that has very low GTPase activity78−80 and the initial constriction of tubular liposomes in the presence of this “GTPase-dead” mutant FtsZ.75 2.6. FtsZ and Cell Morphology. The peptidoglycan layer of bacterial cell walls consists of a heteropolymer of polysaccharides cross-linked with short peptides that function as the load-bearing material to resist mechanical and physical forces (e.g., osmotic pressure) on cells. During the growth of rod-shaped cells, peptidoglycan is assembled in two distinct regions of the cell: (1) along the cylindrical body of cells, which is required for cell elongation, and (2) at the site of cell division, which creates a new curved pole for the two daughter cells. FtsZ is required for septal/cell-division-associated peptidoglycan growth and remodeling due to its essential role in recruiting cell-division-specific peptidoglycan synthesis enzymes.49,50 However, recent research suggests that the regulatory role of FtsZ on peptidoglycan synthesis during cell division extends beyond its ability to recruit proteins to the mid-cell.7 Particularly, it was recently shown that the intrinsically disordered C-terminal linker region of FtsZ is important for regulation of enzymes involved in peptidoglycan metabolism in C. crescentus.7 In addition to septal/cell-division-associated peptidoglycan growth mediated by the divisome, rod-shaped bacteria have other cellular machinery mediating lateral peptidoglycan synthesis along the length of the cell. This multiprotein complex named the “elongasome” is organized by the ancestral homologue of actin, MreB.49 Until recently, the role of FtsZ has been thought to be restricted to participating in peptidoglycan assembly and remodeling at the division site. However, recent studies have demonstrated that FtsZ may also play a role in elongation-associated cell wall growth in rodshape bacteria.6,81,82 The direct role of an FtsZ homologue in cell shape control of rod-shaped microorganisms has been also demonstrated in archaea. Unlike most bacteria, archaeal genomes frequently contain additional genes belonging to the FtsZ/tubulin superfamily.83 The archaeal tubulin-like protein CetZ, formerly annotated as “FtsZ3” or “FtsZ type 2”, has been implicated in cell shape control of Haloferax volcanii.84 CetZ has the FtsZ/ tubulin superfamily fold and a crystal form containing sheets of protofilaments that suggest it may play a structural role in cells. Inactivation of CetZ1 in H. volcanii does not affect cell division; however, it prevents differentiation of the irregular plate-shaped cells into a rod-shaped cell type essential for normal swimming motility. CetZ1 forms dynamic cytoskeletal structures in vivo, indicating its capacity to remodel the cell envelope and direct rod formation.84 6979 DOI: 10.1021/acs.jmedchem.5b01098 J. Med. Chem. 2016, 59, 6975−6998 Journal of Medicinal Chemistry Perspective 3. MECHANISMS OF ACTION OF INHIBITORS OF FtsZ Several characteristics validate FtsZ as a target for the development of new antibiotics to selectively combat bacterial infections: (1) it is essential and plays a specific role in prokaryotic cell division;4 (2) it is structurally and functionally conserved across bacterial and archaeal species;15,29,85 (3) although widespread in mitochondria of diverse protist lineages, it is notably absent in higher eukaryotes;83,86 (4) it is evolutionarily distant from its eukaryotic counterpart tubulin;3,13,31,32 and (5) there is a growing body of research on its structural, biochemical, and biological properties. 3.1. Antagonism of Polymerization and GTPase Activity of FtsZ by Small Molecules. Hydrolysis of GTP requires assembly of two FtsZ monomers to complete the catalytic site. This innate step in catalysis can be modulated by targeting either the T7 loop of the “upper” monomer or the nucleotide-binding pocket of the “lower” monomer.87 Alternatively, an allosteric site on FtsZ may modulate its ability to form protofilaments. In this sense, the tightly regulated division process could be halted by several mechanisms, including (1) overly stabilizing protofilaments, which cannot disassemble as GDP is produced by GTP hydrolysis; (2) destabilizing protofilaments; and (3) preventing polymerization. The chemical inhibitors of FtsZ reported to date can be classified into three main groups: (1) natural products and their derivatives; (2) nucleotide analogs; and (3) molecules that emerged from high-throughput screening. Below, we provide an overview of the inhibitors that were a starting point for further development of structurally related compounds or assays for their activity as inhibitors of cell division. However, a caveat to this list is that many of these inhibitors have been shown to be false positives or to have irreproducible activity. This section delineates the reported, albeit limited, mechanistic detail of reported FtsZ inhibitors, which lays the foundation for a discussion of validated inhibitors in section 4. A summary of the antimicrobial activity, discovery methods, and FtsZ binding characteristics of these compounds is presented in Table 3. 3.1.1. FtsZ Inhibitors from Natural Products: Alkaloids. Sanguinarine (1) is a polycyclic alkaloid that inhibits FtsZ protofilament assembly by decreasing FtsZ polymerization;88 it also inhibits eukaryotic tubulin, which complicates its use as an antibiotic. Berberine (2) is a structurally related alkaloid that inhibits GTPase activity and decreases FtsZ polymerization. It is predicted to bind in the vicinity of the GTP binding pocket and overlaps with several hydrophobic residues located in the GTP binding site.89 Although allegedly indifferent to tubulin, 2 has since been described as a promiscuous binder of different proteins.90 Berberine 2 (3) was designed to have an extended alkyl group in place of one of the methyl groups on 2; the in vitro GTPase inhibition activity of 3 was measured to be approximately 38 μM against S. aureus FtsZ.91 3.1.2. FtsZ Inhibitors from Natural Products: Polyphenols. Plumbagin (4) inhibits the GTPase activity of FtsZ and increases the lag phase of FtsZ assembly (i.e., adversely affects the nucleation rate). The predicted binding site of 4 is located close to the C-terminal domain of FtsZ in a region of the H7 helix, spatially distant from the GTP binding domain.92 SA-011 (5)93 was synthesized as an analog of 4 and shown to inhibit the GTPase activity of Bacillus anthracis slightly better than 2. Resveratrol (6) has been screened many times due to its known antimicrobial activity, which has been attributed to inhibiting Z-ring formation and suppressing the expression of FtsZ mRNA.92,94 Dichamanetin (7) and 2‴-hydroxy-5″-benzylisouvarinol-B (8) are structurally similar pinocembrin-based molecules that inhibit the GTPase activity of FtsZ in Gram-positive bacteria. 8 also displays antimicrobial activity against E. coli and Pseudomonas aeruginosa.95 However, 7 was later shown to be an “aggregator”, a molecule that forms aggregates that bind nonspecifically to proteins. Viriditoxin (9) was initially reported to inhibit the GTPase activity of FtsZ and cause cells to filament, while overexpression of FtsZ was shown to rescue drug-treated cells.96 9 has since been confirmed as a falsepositive that has activity that has not been reproducible.97 The complex natural product family of chrysophaentins (e.g., chrysophaentin A (10)) was shown to inhibit the GTPase activity of E. coli and S. aureus FtsZ (including methicillinresistant S. aureus strains), and molecular docking experiments showed that the compound occludes a large portion of the GTP binding site of the protein.98 FtsZ polymerization is inhibited, and the Z-ring is mislocalized in cells treated with 10. Despite these results, cell filamentation was not observed in a mutant strain of E. coli (envA1) permeable to a wide variety of compounds.99 3.1.3. FtsZ Inhibitors from Natural Products: Phenylpropanoids and Terpenoids. Several phenylpropanoids that are derived from cinnamaldehyde (11) or related structures have been tested for antimicrobial activity. Nearly all phenylpropanoids described as FtsZ inhibitors to date are alleged to interact with at least one residue of the T7 loop.100 Virtual screens and/or docking experiments of many of these structurally “simple” natural products suggest they have specific interactions with FtsZ. However, few examples have translated into reliable inhibitors and lack biophysical support for targeting FtsZ. 11 inhibits the GTPase activity of FtsZ, decreases polymerization and is not toxic to red blood cells.101 Phenylacrylamide 14 (12)102 has antibacterial activity against S. aureus and Streptococcus pyogenes and inhibited cell division in S. aureus. Vanillin derivatives 3a (13)103 and 4u (14)104 have been independently tested against Mycobacterium tuberculosis FtsZ. Scopoletin (15), a coumarin analog related to esculetin and quercetin, inhibits the GTPase activity and polymerization of FtsZ into protofilaments.105 Curcumin (16) increases the GTPase activity and destabilizes polymerization of FtsZ, thus reducing the steady-state duration of polymer assembly.106 Unlike other phenylpropanoids, the predicted FtsZ binding site of 16 involves residues connected to GTP binding of FtsZ.107 Colchicine (17), although highly active against tubulin polymerization, has also been tested against FtsZ and has been demonstrated to have no effect on FtsZ polymerization.108 Sulfoalkylresorcinol (18) inhibits the GTPase activity of FtsZ in vitro and exhibits antimicrobial activity against various pathogens but is not cytotoxic toward human A549 cells.109 Synthetic derivative n-undecyl gallate (19) disrupts ZapA localization and possibly Z-ring formation in Xanthomonas citri subsp. citri.110 Totarol (20) is a terpenoid that inhibits the GTPase activity and polymerization of FtsZ protofilaments. Cells treated with 20 become filamentous and display a mislocalized Z-ring.111 20 was initially described as lacking activity against eukaryotic tubulin; more recently it has been shown to be promiscuous in binding to proteins and to have properties consistent with being an aggregator.97 Germacrene D (21) and germacrene D-4-ol (22) are part of a family of terpenoids isolated from the essential oil of pine needles that exhibit antibacterial activity on 6980 DOI: 10.1021/acs.jmedchem.5b01098 J. Med. Chem. 2016, 59, 6975−6998 Journal of Medicinal Chemistry Perspective Table 3. Summary of Reported FtsZ Inhibitors Discussed in the Texta 6981 DOI: 10.1021/acs.jmedchem.5b01098 J. Med. Chem. 2016, 59, 6975−6998 Journal of Medicinal Chemistry Perspective Table 3. continued 6982 DOI: 10.1021/acs.jmedchem.5b01098 J. Med. Chem. 2016, 59, 6975−6998 Journal of Medicinal Chemistry Perspective Table 3. continued 6983 DOI: 10.1021/acs.jmedchem.5b01098 J. Med. Chem. 2016, 59, 6975−6998 Journal of Medicinal Chemistry Perspective Table 3. continued a Ec = Escherichia coli; EcFtsZ = recombinant E. coli FtsZ; SaFtsZ = recombinant Staphylococcus aureus FtsZ; BsFtsZ = recombinant Bacillus subtilis FtsZ; MtFtZ = recombinant Mycobacterium tuberculosis FtsZ; PaFtsZ = recombinant Pseudomonas aeruginosa FtsZ; BaFtsZ = recombinant Bacillus anthracis FtsZ; MRSA = methicillin-resistant Staphylococcus aureus core and altered oxidation pattern (denoted as TRA 10a (25) or 10b (26)) improved the potency of this taxane-derived structure to target M. tuberculosis FtsZ.114 3.1.5. FtsZ Inhibitors That Mimic Nucleosides. Nucleotide analogs have been explored as competitive inhibitors of GTP for binding to FtsZ. 8-Bromoguanosine 5′-triphosphate (27) binds to FtsZ with a Ki of 32 μM and inhibits both FtsZ polymerization and GTPase activity.115 Gal cores 10 (28), 14 (29), and 15 (30)116 were designed to mimic the sugar-phosphate backbone of GTP and shown to inhibit the GTPase activity of P. aeruginosa FtsZ through an enzyme-coupled assay. 3.1.6. FtsZ Inhibitors from High-Throughput Screening of Chemical Libraries. A number of structurally distinct small molecules emerged from in vivo high-throughput screens as causing cell filamentation and were considered to be targeting FtsZ. Quinoline 1 (31)117 was screened against M. tuberculosis various species of bacteria. A docking model predicts a binding site of the germacrene family to be a hydrophobic pocket in FtsZ; however, the crystallographic evidence for this interaction is not yet determined.112 3.1.4. FtsZ Inhibitors That Are Derived from Taxanes. Taxane-derived structures have been successfully modified to target FtsZ preferentially over its eukaryotic homologue tubulin. The taxane polycyclic core in these compounds has remained largely intact. For example, SB-RA-2001 (23)113 only differs from paclitaxel (24) in two ways: (1) the alcohol at C-10 lacks an acetyl group, and (2) an unsaturated ester has replaced the α-hydroxy-β-amido ester of 24 at C-13. These structural changes led to inhibition of enzymatic activity against B. subtilis FtsZ and were shown to have antimicrobial activity against both B. subtilis and Mycobacterium smegmatis. Huang et al. later showed that the conjugated ester coupled with a ring-opened 6984 DOI: 10.1021/acs.jmedchem.5b01098 J. Med. Chem. 2016, 59, 6975−6998 Journal of Medicinal Chemistry Perspective Compound 297F (49)135 is the most structurally divergent compound in the benzamide family of FtsZ inhibitors and targets M. tuberculosis; however, it bears little structural resemblance to 42. 3.1.8. FtsZ Inhibitors Based on a Benzimidazole Scaffold. A series of novel trisubstituted benzimidazoles, which were inspired by tubulin-targeting thiabendazole (50) and albendazole (51),136 have been reported as targeting M. tuberculosis FtsZ (Mtb-FtsZ). 137−142 Two benzaimidazoles, 1a-G7 (SB-P3G2) (52) and 1a-G4 (53),137 were shown to enhance the GTPase activity of Mtb-FtsZ but inhibit the Mtb-FtsZ polymerization in a dose-dependent manner. 5f (SB-P17G-C2) (54) and 7c-4 (SB-P17G-A20) (55)138 were identified as potent bactericidal benzimidazoles in SAR studies. These benzimidazoles did not show significant cytotoxicity against a VERO eukaryotic cell line.137,138 Those potent benzimidazoles effectively inhibited the polymerization of Mtb-FtsZ and also caused the depolymerization of existing Mtb-Ftz protofilaments.138 52 and 55140,141 showed efficacy in acute tuberculosis model studies in mice. Optimized analogs of this series of compounds, SB-P17G-A38 (56) and SB-P17G-A42 (57),142 were recently reported to have efficacy in a tuberculosis infection animal model. 3.1.9. FtsZ Inhibitors That Incorporate Other Heterocyclic Scaffolds. Quinuclidines 1 (58)143 and 12 (59)144 differ by a hydroxyl or N-methylamino group, respectively. Both compounds inhibit growth of a variety of bacterial species and have broad-spectrum activity. The quinuclidine core was suggested to bind to the GTP pocket of FtsZ through docking models of the M. jannaschii crystal structure (PDB code 1W5B). Fluorophores such as DAPI (60) have been shown to inhibit the GTPase activity of FtsZ but do not affect polymerization of tubulin.145 4-Bromo-1H-indazole 12 (61)146 was designed from the charged alkaloids 1 and chelerythrine (62). 61 showed moderate antibacterial activity with an MIC of at least 128 μM against any species of bacteria tested. 5,5-Bis-8-anilino-1naphthalenesulfonate (63) inhibits the binding of GTP to FtsZ and is significantly affected by the concentration of calcium ions present. The concentration of calcium ions also induces conformational changes of FtsZ and might thus be the more important effect to consider in modulating the activity of this protein.108 3.2. Altering FtsZ Activity or Stability by Targeting FtsZ Regulators. Membrane−protein and protein−protein interactions are critical for assembly of the divisome (Figure 2A and Figure 2B). As described in section 2, proteins that modulate FtsZ synthesis, polymerization, activity, and turnover are essential for ensuring the precise spatiotemporal regulation of cytokinesis. In principle, many of these factors can be explored as potential targets for the development of FtsZ inhibitors (Table 2), and yet this area is largely unexplored. In the next section we describe three examples of general, conserved FtsZ regulators that can be explored as potential indirect targets of FtsZ. 3.2.1. Altering FtsZ Activity by Disrupting the ZipA−FtsZ Interaction. In E. coli and other γ-proteobacteria, the transmembrane protein ZipA is one of the essential components of the divisome responsible for recruitment of FtsZ to the membrane (Table 2).36,50,69 ZipA binds specifically to residues confined to the C-terminal region of FtsZ.36 There are at least two examples in the literature of groups of small molecules that disrupt the interactions between ZipA and FtsZ. The indolo[2,3a]quinolizin-7-one inhibitors (compounds 1 (64) and 10b (65)) and selected for its antibacterial potency and selectivity for bacteria over mammalian cells. 31 was hypothesized to bind to the putative colchicine pocket of M. tuberculosis FtsZ based on chemoinformatics modeling. Rhodanines (e.g., OTBA (32)) increase protofilament assembly/bundling and inhibit GTPase activity of FtsZ but do not affect the secondary structure of FtsZ. 32, not surprisingly, also inhibits the proliferation of HeLa cells118 and generally affects many proteins nonselectively, as evidenced by its proliferation as a “PAINs compound” in high-throughput screens (vida infra). Aminofurazan A189 (33)119 was identified in a chromosome partitioning screen by an anucleate cell blue assay that looked specifically for the inhibition of GTPase activity of FtsZ, although no binding site was predicted for the compound with either E. coli or S. aureus FtsZ. Aminopyridines SRI-3072 (34) and SRI-7614 (35)120 inhibit the GTPase activity of M. tuberculosis FtsZ and reduced bacterial growth in mouse bone marrow macrophages. UCM44 (36)121 inhibits the GTPase activity of FtsZ in B. subtilis but only marginally for E. coli FtsZ. A family of structurally unrelated small molecules referred to as the “zantrins” were reported to affect FtsZ protofilament assembly and inhibit GTPase activity; zantrins Z1 (37), Z2 (38), and Z4 (39) decreased the length of FtsZ protofilaments, and zantrins Z3 (40) and Z5 (41) stabilized FtsZ protofilaments.122 37 and its chemical relative trisphenol 7 were further pursued due to their shared structural scaffolds but were found to be small molecules aggregators and not bona fide inhibitors of FtsZ. Due to their poor prospects as drug leads, 38, 39, and 41 were not examined in subsequent studies. Recent SAR studies of 40 demonstrated that a substituted quinazoline ring could retain the potency of the parent compound and incorporation of a small, positively charged side chain improved activity by 3-fold.123 3.1.7. FtsZ Inhibitors Based on a Benzamide Scaffold. Studies of the benzamide family of small molecules over the past 15 years culminated in the development of PC190723 (42)124 from the starting inhibitor 3-methoxybenzamide (43).125 42 was the first non-nucleotide inhibitor of FtsZ to be cocrystallized with FtsZ.126 42 was initially described as stabilizing FtsZ protofilaments127 and inhibiting GTPase activity;124 however two independent groups later demonstrated that the compound decreases the cooperativity of FtsZ monomers,130 activates the GTPase activity of S. aureus FtsZ,97,128 and resensitizes MRSA to β-lactams.126 The cocrystal structure of FtsZ and 42 is consistent with binding of this molecule between strand 8 and helix H7, which disrupts the conserved hydrogen bonds that enable helix H7 to communicate with the GTP binding site. This translocation of helix H7 decreases the lag phase of FtsZ polymerization, altering the cooperativity of the FtsZ monomers.126,128 42 mislocalizes the Z-ring in S. aureus cells; however it does not disrupt the division proteins that localize to the FtsZ foci.129 Although limited to S. aureus, 42 is currently the best inhibitor of FtsZ to date and is a useful tool for microbiology. However, poor solubility and formulation properties have hindered 42 from clinical use. 42 has been modified into various prodrugs such as TXY436 (44)130 to improve its poor oral bioavailability. 44 and 42 have been further developed into the metabolically more stable analog TXA709 (45), in which the chlorine substituent is replaced with a trifluoromethyl group;131 preclinical studies of this compound are in progress.132 Related benzamide 8J (46)129 is structurally similar to 42 and varies only at the benzothiazole ring system, while (R)-13 (47)133 and compound 1 (48)134 contain the 3-alkoxy-2,6-difluorobenzamide core. 6985 DOI: 10.1021/acs.jmedchem.5b01098 J. Med. Chem. 2016, 59, 6975−6998 Journal of Medicinal Chemistry Perspective division by binding to and sequestering monomeric FtsZ and reducing the effective concentration of FtsZ in cells.169 SulA is highly conserved among Enterobacteriaceae,170 and its role as a potent inhibitor of bacterial cell division could be exploited as a potential target for the development of antibiotics that inhibit cell division by modulating FtsZ. 3.3. Inhibiting FtsZ by Disrupting the Cellular Transmembrane Potential in Bacteria. Membrane potential (ΔΨ) is essential for proper subcellular localization of some cell-division-related proteins, such as MinD and FtsA in B. subtilis and E. coli.171 Consistent with this model, ionophores (e.g., CCCP and valinomycin) and bacteriocins (e.g., nisin and colicin N) that cause depolarization of the cell membrane abolish the oscillation of MinD and the mid-cell localization of FtsA. The detailed mechanism underlying ΔΨ-dependent localization of these membrane proteins is not completely understood, but in vitro experiments suggest that, at least for MinD, the membrane potential stimulates the interaction between the C-terminal amphipathic helix of MinD and the phospholipid bilayer.171 A reduction in mid-cell localization of FtsZ and ZapA occurs after treating cells with CCCP and is correlated with FtsA mislocalization,171 which is important for Z-ring stabilization at the membrane. This finding confirms that inhibition of other proteins or cellular components that interact with FtsZ and regulate FtsZ dynamics can be explored as potential targets for altering FtsZ activity. Many FtsZ inhibitors including 11, 1, 20, 9, and 37 affect the oscillatory behavior of MinD by reducing the membrane potential and affecting membrane permeability.172 As discussed in section 4.6.1, some of the compounds classified as FtsZ inhibitors in vitro do not cause cell filamentation, one of the phenotypic hallmarks of FtsZ inhibition in vivo. These observations suggest that the activity of these compounds on FtsZ may arise as the downstream consequence of their effect on bacterial membranes. 3.4. Inhibition of FtsZ Synthesis Using Short Antisense Oligoribonucleotides. The finding that the endonuclease ribonuclease P, essential for maturation of the 5′ end of tRNAs, can be used to digest target RNA molecules upon addition of an appropriate complementary oligoribonucleotide led to the development of EGS technology.173 The ability to interfere with f tsZ gene expression has been recently investigated as an alternative therapeutic strategy to block bacterial cell division.174 Expression of an EGS targeting the f tsZ mRNA induces cell filamentation and causes growth inhibition in E. coli cells. EGS techniques are still at an early stage of development; however they have been used as antibacterial agents and to inhibit expression of resistance genes in bacteria.175 In principle, EGS approaches could be an efficient strategy to overcome the increase of multiresistance among bacterial pathogens. were shown to affect ZipA−FtsZ interaction by occupying a hydrophobic cavity on the surface of ZipA necessary for the binding to FtsZ. Consistent with this model, in vitro studies showed that various analogs were able to inhibit binding of ZipA to a small peptide that mimics the C-terminal 16 amino acid residues of E. coli FtsZ.147 Similarly, a structure-based study of carboxybiphenylindole inhibitors (e.g., compound 14 (66)) demonstrated binding of a small peptide that mimics the C-terminal 16 amino acid residues of FtsZ and C-terminal domain (residues 185−328) of ZipA.148 3.2.2. Modulating FtsZ Stability through Degradation by the ClpXP Protease. ClpXP is a two-component ATPdependent bacterial protease that controls protein turnover by proteolysis.149 The substrate recognition domain of ClpXP (the ClpX chaperone) can function in a ClpP-independent manner preventing protein assembly and aggregation or remodeling and disassembling macromolecular complexes/ aggregates.150−152 In E. coli and in B. subtilis, the ClpX chaperone inhibits formation of the Z-ring in a ClpPindependent fashion through a mechanism that does not require hydrolysis of ATP, suggesting that ClpX physically blocks the assembly of FtsZ protofilaments.153−155 Similarly, ClpX regulates Z-ring assembly in M. tuberculosis by interacting with FtsZ. Consistent with the model, overexpression of clpX inhibits Z-ring assembly and reduces viability of M. tuberculosis.156 Genetic and biochemical studies in E. coli have shown that the two-component ClpXP protease modulates the dynamics of FtsZ filaments via degradation of FtsZ monomers and protofilaments.38 Bacterial cells overproducing ClpX or ClpXP arrest cell division and have a filamentous morphology.38,153,155 These observations demonstrate that the possible specific activation of the proteolytic activity of ClpXP affecting the stability of FtsZ could be an avenue for FtsZ inhibition and potential antibacterial agent development. A new approach to inhibit cell division through FtsZ by targeting the bacterial proteolytic machinery was demonstrated recently.157−159 Acyldepsipeptides (ADEPs, 67−71) are natural product-derived antibiotics active against Gram-positive bacteria, and their mechanism of action involves uncontrolled proteolysis of FtsZ mediated by ClpP peptidase.159 Biochemical and structural data showed that this family of compounds competes with the Clp ATPases for the same binding site, stimulates ClpP activity through cooperative binding, and induces uncontrolled ClpP-dependent proteolysis, decreasing the abundance of FtsZ and inhibiting cell division.157−160 It is not clear why FtsZ is particularly sensitive to ADEP-ClpP; however the mechanism is dependent on the structure of the protein, as α- and β-tubulins are also targets of the ADEP-ClpP complex.159 3.2.3. Inhibiting FtsZ by Activating the SOS Pathway. Following DNA damage, E. coli and related Gram-negative bacteria activate an elaborate cellular program (the SOS response) for DNA repair and cell survival.161 The division inhibitor SulA is synthesized as part of the SOS response,162,163 which causes cell filamentation by inhibiting polymerization of FtsZ at the division site.164−166 After DNA is repaired, SulAmediated inhibition of FtsZ is rapidly released by proteolysis of SulA,167 restoring the ability of FtsZ to polymerize and re-form the Z-ring. SulA binds FtsZ monomers in a 1:1 ratio, and GTP is required for SulA binding in vitro.165,168 Although there is evidence that SulA inhibits the GTPase activity of FtsZ,165,168 a recent study suggests that SulA inhibits cell 4. STRATEGIES FOR DISCOVERING NEW FtsZ INHIBITORS 4.1. High-Throughput Screening. Target-based (largely in vitro) and whole-cell (in vivo) high-throughput screens have been used to identify FtsZ small molecule inhibitors. The majority of target-based approaches require recombinant, purified protein and do not select for compounds that have favorable transport properties across bacterial membranes.176 The most common screens of FtsZ inhibitors have assayed inhibition of the GTPase activity or FtsZ polymerization in the presence of a small molecule.118,122,135 Other in vitro screens 6986 DOI: 10.1021/acs.jmedchem.5b01098 J. Med. Chem. 2016, 59, 6975−6998 Journal of Medicinal Chemistry Perspective were not discovered in screens per se. Examples include 11,101 15,105 16,106 2,184 1,88 4,92 and 20111 (Table 3). Several of these compounds have off-target effects185 and challenges with aggregation.97 4.3. Virtual Screening Using the FtsZ Crystal Structure. A virtual screening approach utilizes known structural data of a prioritized target and identifies or optimizes a ligand for a binding site of interest. Ligands can be chemical structures extracted from databases or built from chemical fragments docked into regions of the binding site.186 Databases are constructed to include chemical structures with a high degree of druglikeness with physically relevant conformations.187 Virtual screening using crystal structures of FtsZ have identified new inhibitors. Specifically, inhibitors of the GTPase activity of FtsZ have been predicted by docking compounds into the GTP binding site of FtsZ. One virtual screen focused on docking compounds from natural product collections,144 while another screen utilized known scaffolds from the literature, an in-house synthetic library of small molecules, and 4 000 000 compounds from a virtual library.121 Both of these approaches identified compounds that are structurally distinct from GTP and had moderate activity against FtsZ in vitro. A select number of these compounds demonstrated moderate antibacterial activity against Gram-positive organisms,121 confirming the value of this discovery approach. 4.4. Molecular Modeling and SAR Studies To Identify FtsZ Inhibitors. Molecular modeling using a protein crystal structure is often used to optimize hits from a high-throughput screen to improve binding and activity. This strategy decreases the cost and amount of time for SAR studies and provides compounds for biochemical studies and further protein crystallization. A new pharmocophore that inhibits the interaction between FtsA and ZipA was identified in a highthroughput screen and cocrystallized with FtsZ.178,179 By use of the shape-comparison program ROCS, new scaffolds were identified that have potential for synthetic optimization.188 Rational design using molecular modeling is one possible strategy when a protein crystal structure is available and docking experiments can be performed. For example, rational design of FtsZ inhibitors using molecular modeling methods can leverage molecules that mimic GTP, such as 27, compete with GTP for binding to FtsZ, and inhibit the GTPase activity (Table 3). An analysis of GTP bound to FtsZ in the crystal structure reveals positions on the nucleotide that can be modified to obstruct the binding of GTP to FtsZ.115 Docking experiments with 2 (Table 3) and FtsZ predicted that 2 binds to the C-terminal cleft near the GTP binding site and identified the C9-methoxy position of 2 as a possible locus for modification to optimize its activity.91 Another approach to inhibit FtsZ function is to use modeling to target and disrupt the FtsZ interface with proteins that localize it and affect its function. For example, docking experiments were used to optimize the structure of 66 to disrupt the ZipA− FtsZ interaction148 (Table 3). SAR studies can also lead to potent analogs without structural or computational guidance. By use of combinatorial chemistry, GTP analogs with two side chain substitutions (compounds 28, 29, and 30) in the place of the ribose and the triphosphate were synthesized and tested for inhibition of the GTPase activity of FtsZ116 (Table 3). An amine modification library of 64 (an inhibitor from the study mentioned previously148) resulted in a moderate increase in the inhibition have measured the small molecule inhibition of the interaction between FtsZ and ZipA using fluorescence polarization.177,178 A challenge with in vitro assays of recombinant proteins is that they often yield inhibitors that are not very active in vivo. Challenges with cell uptake and bioavailability (e.g., drug efflux) of inhibitors developed through in vitro high-throughput approaches have created new momentum for whole-cell chemical screens.176 Most whole-cell, high-throughput assays have been designed to identify compounds that inhibit cell growth rather than to focus on a specific antibacterial target. This strategy serendipitously led to the identification of the quinolone and quinazoline families of FtsZ inhibitors.117 Whole-cell strategies have been developed to identify FtsZ inhibitors.121,174,175,179 A clever example of a cell-based reporter assay for FtsZ utilized the activation of the σF transcription factor during sporulation in B. subtilis as a reporter of septum formation at the site of cell division. This dual reporter system monitors the expression levels of promoter-fusions for the σF-dependent spoIIQ promoter and spoIIA promoter, which is only active before septation. In the presence of a cell division inhibitor, the concentration of spoIIQ decreases and spoIIA increases, which inhibits septum formation. The assay has a built-in control to reduce nonspecific inhibitors, which cause the levels of spoIIQ and spoIIA to decrease.180 Once a hit is identified in whole-cell assays, secondary assays are used to confirm FtsZ as the target. A growing consensus is that engineering cell permeability into a lead compound identified from a screen is more difficult than developing in vivo assays, and consequently whole-cell assays are being used frequently for high-throughput screening.176 4.2. Screening Natural Product Libraries and Extracts for FtsZ Inhibitors. Libraries of synthetic small molecules have been widely assayed in high-throughput screens to identify FtsZ inhibitors. An increasing number of screens have focused on natural products. Natural products were the primary source of antimicrobial compounds in the early years of antibiotic discovery and comprise 77% of the antibiotics used clinically since 2000 (all of which were derived from microbes).181 A decrease in the rate of antibiotic discovery, among other factors, shifted attention from natural products to semisynthetic natural products and entirely synthetic compounds. One key challenge of antibiotic discovery is that the most successful antibiotics often do not follow Lipinski’s rules of druglike compounds. Consequently, libraries of synthetic compounds (which are curated for compounds that follow Lipinski’s rules) are missing key chemical space for antibiotic drug discovery.176 Natural products are intrinsically biologically active, and compounds have been evolutionarily selected for their transport properties into bacterial cells.182 These and other properties could fill this missing chemical space found in chemical libraries. The development and maintenance of natural product extract libraries have emerged as a method of increasing the chemical diversity of compound libraries for antibiotic drug discovery.183 High-throughput assays have been used to screen natural product extract libraries for FtsZ inhibitors in vitro.93,96 Also, virtual structural libraries of natural products and their semisynthetic derivatives have been implemented using in silico high-throughput assays.144 Preliminary whole-cell antibacterial activity screening of natural extracts from different sources, such as marine fungi,109 marine alga,98 or certain pine trees,112 have provided new chemical scaffolds that target FtsZ. Many natural products identified as FtsZ inhibitors in vitro 6987 DOI: 10.1021/acs.jmedchem.5b01098 J. Med. Chem. 2016, 59, 6975−6998 Journal of Medicinal Chemistry Perspective of the interaction between FtsZ and ZipA.147 The discovery of 42 is a successful SAR story that did not rely on in silico strategies (Table 3). By use of 43 as a starting point, 500 analogs were synthesized and tested against bacteria, using cell shape to assess biological activity.124 Substitutions on the phenyl ring established that fluorine atoms at the R4 and R7 positions improved antibacterial activity. Compounds with alkyl chains of various lengths at the 3-methoxy position were >10 000 times more potent than 43.189 Further SAR studies improved the druglike ADME properties of the 2,6-difluoro-3alkyloxybenzamides. The replacement of the alkyl chains with heterocycles, such as thiazolopyridines and benzothiazoles, decreased the log P and potential plasma protein binding. The benzothiazole derivative retained its antibacterial activity while lowering the log P value compared to the alkyloxybenzamide derivatives. Analogs with substitutions at each of the available positions on the benzothiazole ring were prepared and tested. Incorporating a chlorine atom at the 5-position and replacing a nitrogen atom at the 7-position of the benzothiazole produced a low log P value, decreased plasma protein binding, and retained potent antibacterial activity against S. aureus.190 Not only does this example demonstrate a successful SAR study of an FtsZ inhibitor, but it also highlights a constant goal in medicinal chemistry to exploit “ligand efficiency.” This term has emerged to describe the level of activity of an inhibitor on a per atom or per dalton basis, which provides a metric for measuring improved potency without sacrificing druglikeness.191 4.5. Screening and Modification of Tubulin Inhibitors. Many potent and selective inhibitors of tubulin originate from natural products and have gone on to become clinical drugs and or drug leads, including 24, vinca alkaloids (vinblastine, vincristine), 17, epothilone, peloruside, maytansine, and halichondrin (the progenitor of eribulin/halaven). Structural biology data have been determined for most of these protein− small molecule complexes.192−195 The contrast of inhibitors of tubulin and FtsZ is stark: there are far fewer natural products that inhibit the function of FtsZ, none of these inhibitors have the potency of the tubulin-targeting molecules listed above, and only one cocrystal structure has been solved to date (FtsZ bound to the synthetic small molecule 42). The origin of this difference may reflect a research bias (e.g., a larger allocation of federal funding to support eukaryotic cell biology), a difference in the susceptibility of each protein to small molecule binders, and/or differences in resistance mechanisms in eukaryotes versus prokaryotes. Vinca alkaloids and taxanes are drugs used for clinical cancer chemotherapy by either destabilizing (vinca alkaloids) or stabilizing microtubules (taxanes). Both activities prevent normal microtubule function in cells. Some vinca alkaloids are FDA-approved (such as vinblastine, vincristine, and vinorelbine) for treating specific types of cancer including lymphomas, sarcoma, leukemias, and non-small-cell lung cancers. FDA-approved taxanes include docetaxel, 24, and nab-24 for treating different types of cancer including breast, gastric, head and neck, prostate, ovarian, and non-small-cell lung cancers. Derivatives of epothilones are currently in clinical development because of their increased potency compared to taxanes and their application in combination drug therapy.196 Tubulin has been a validated cancer treatment target for many years and is structurally similar to FtsZ, as we mentioned briefly in section 2.1 and highlight in Figure 1. Phylogenetic analyses reinforce an evolutionary linkage between FtsZ and eukaryotic tubulin.13,32,197 In fact, FtsZ and tubulin share many essential functional and structural properties, including cooperative assembly stimulated by GTP and dynamic polymerization. The structural homology of Sus scrofa tubulin and M. jannaschii FtsZ is high, and the common core is superimposable with a root-mean-square deviation of 4.3 Å (Figure 1).13 Despite excellent structural homology, distinctions between the two proteins are significant enough that they may be exploited to create chemical inhibitors specific for FtsZ. Loops connecting strands and helices of tubulin are longer than those in FtsZ, causing tubulin to have a wider crosssection than a molecule of FtsZ. A sequence alignment of tubulin and FtsZ demonstrates that the proteins share 7% homology of amino acids, most of which are located in regions associated with nucleotide binding.13 After polymerization in vitro, FtsZ protofilaments associate laterally, which is different from the association of tubulin protofilaments in microtubules (Figure 1). Sheets of FtsZ protofilaments do not have a standard tubulin microtubule lattice. Another physical distinction between the two proteins is that FtsZ minirings consisting of protofilaments are approximately half the diameter of tubulin rings.41 Although the two proteins are structurally homologous, the protofilament bundling arrangement and amino acid sequence of tubulin and FtsZ are distinct, which provides a platform for target specificity. A challenge of FtsZ inhibitor discovery is to identify molecules that do not target eukaryotic tubulin, a step referred to as the “antitubulin approach”. Many of the classic tubulin inhibitors do not have significant activity against GTPase activity and polymerization of FtsZ, demonstrating that target specificity is possible. Examples of this specificity are the tubulin inhibitors 17 and 51, both of which show no significant activity against FtsZ polymerization and GTPase activity.16 Similarly, the cross-species activity of an inhibitor can in principle be finetuned to target only FtsZ. 34 and 35 were identified as M. tuberculosis FtsZ inhibitors from a synthetic tubulin inhibitor library120 (Table 3). An SAR study enhanced the antibacterial activity of the compounds and increased the specificity for inhibition of FtsZ over tubulin.198 The benzimidazole scaffold was picked out of the same tubulin inhibitor library as a promising antagonist of FtsZ (Table 3), and a library of 2,5,6- and 2,5,7-trisubstituted benzimidazoles were synthesized and tested against drug-sensitive and drugresistant M. tuberculosis. A cyclohexyl group at the 2-position was preferred and the results motivated the investigation of substitutions at the 5- and 6-position of the benzimidazoles,137 which led to two potent analogs with a dimethylamino group at the 6-position and a benzamide or a carbamate at the 5-position.138 Additionally, the investigation of the 6-position was expanded to create a library of trisubstituted benzimidazoles with ether and thioether substituents at the 6-position; however none of the 6-ether/thioether analogs were as potent as the analogs with the dimethylamino group at the 6-position.199 This SAR study is an excellent example of rational drug design using tubulin inhibitors as a starting point to discover new molecules with specificity for inhibiting FtsZ. 4.6. Strategies To Improve Characterization Methods of FtsZ Inhibitors. Chemical hits against FtsZ from in vivo whole-cell screens or natural product extracts are typically confirmed using in vitro experiments, such as GTP hydrolysis and protofilament assembly. Conversely, hits from in vitro highthroughput or virtual screens are tested in vivo for phenotypic traits of FtsZ inhibitors, such as cell filamentation or subcellular localization of the Z-ring.24 6988 DOI: 10.1021/acs.jmedchem.5b01098 J. Med. Chem. 2016, 59, 6975−6998 Journal of Medicinal Chemistry Perspective A drug whose dose−response curve deviates from the standard sigmoidal shape and instead forms a bell-shaped curve at higher concentrations of drug should be scrutinized as a potential aggregating molecule.205 One method for detecting the presence of drug aggregates is implementing a detergentbased control using Triton X-100, which dissolves drug−drug aggregates.206 However, Triton X-100 has been shown to disrupt the protein−protein interactions between the tubulin polymers as evidenced by electron microscopy.121 Nevertheless, a cross-comparison of Triton X-100 versus centrifugation or increasing protein concentration showed similar results for 37, 42, and 20.97 Furthermore, Triton X-100 has been used in control experiments with derivatives of 24113 and analogs of 2.91 Bona fide inhibitors of FtsZ will produce the same dose− response curve with or without an aggregation test, while an aggregator will result in a loss of activity at any drug concentration. In vitro measurements of GTP hydrolysis by FtsZ are complicated by the complexity of the biological coupling between GTPase activity and FtsZ protofilament formation. A thorough reexamination of several reported FtsZ inhibitors indicates two are aggregators (e.g., 7 and 37); one is a PAINs compound (e.g., 32), and several inhibitors have activity that vary significantly from earlier reports (e.g., 9).97 Aggregation is a problem for in vitro assays, such as GTPase activity measurements and light scattering experiments, in which a nonspecific aggregation effect of small molecule inhibitors with FtsZ monomers impedes polymerization. FtsZ protofilaments and bundles can be observed by electron microscopy; however this technique is unable to decouple whether a decreasing FtsZ polymerization rate and the change in the number of protofilaments are a result of aggregation or an inhibitor binding to FtsZ specifically. FRET assays can be used to transcend this limitation. One population of FtsZ proteins can be labeled with a donor fluorophore (a FRET donor) and another with an acceptor fluorophore (a FRET acceptor). Polymerization of FtsZ monomers creates a spatial distribution of fluorophores; a donor and acceptor positioned adjacent to each other will produce a FRET signal.207 An inhibitor that disrupts FtsZ polymerization may reduce the FRET signal. Cell filamentation arises in response to alterations in the topological state or structure of DNA. As mentioned in section 3.2.3, when the SOS response is triggered in response to DNA damage, SulA inhibits FtsZ polymerization (Table 2) and cell filamentation occurs. However, bacteria can filament via SulA-independent mechanisms after the SOS response is triggered by DNA damage, perturbation of DNA topology, and stalling of the DNA replication fork.185,208 The first step in the SOS response is controlled by the RecA-activated selfcleavage of LexA, a transcriptional repressor of all the SOS response genes.208 Therefore, the use of a ΔsulA mutant with a noncleavable LexA repressor (lexA (ind−)) is more informative than a ΔsulA mutant, as the activation of all of the SOS response genes and their contribution to SulA-independent filamentation should be assessed. As referred above in section 2.2, the depletion of other Fts proteins yields filamentous cells that are multinucleate (i.e., they have multiple copies of chromosomes that are evenly spaced along the length of the filament),209 suggesting that inhibiting Fts proteins (other than FtsZ) produces a filamentous phenotype. Inhibiting some clinically relevant antibacterial targets (such as inhibitors of peptidoglycan biosynthesis and DNA supercoiling) produces a filamentous cell phenotype. The in vitro measurement of GTP hydrolysis activity has become a standard assay for FtsZ inhibitors. As mentioned earlier, target-based high-throughput screens were designed to monitor GTPase activity by measuring the release of inorganic phosphate using a malachite green dye assay99,106,111,118,135,137 or coupled enzyme assays.122,200 A common secondary assay to evaluate compounds in vitro is monitoring the inhibition or stabilization of FtsZ protofilaments and bundles using light scattering or electron microscopy.88,89,101,105,106,115,118,127,135,137,179,201 Further in vitro studies can be performed to characterize FtsZ inhibition by evaluating the intrinsic properties of FtsZ. The conformational changes of FtsZ and its bundles in the presence of inhibitor are measurable by far-UV circular dichroism.100,105,106,118 Many assays have been developed to demonstrate compounds binding to FtsZ directly. A simple method is to perform a sedimentation assay that takes advantage of the formation of insoluble protofilaments as FtsZ polymerizes, which precipitate out of solution and can be collected by centrifugation. If an inhibitor prevents polymerization, the amount of sedimented FtsZ decreases; conversely, the amount of sedimented FtsZ increases if the inhibitor stabilizes protofilaments.111,118,122,127,135,179 Several examples of competitive binding assays have been reported that use fluorescent inhibitors,106,108 fluorescent probes,111 fluorescent GTP nonhydrolyzable analogs,99,111,179 and modification of FtsZ with fluorescein96 or tryptophan residues.92,118,179 All of these in vitro techniques facilitate characterizing compounds as GTPase inhibitors or activators that disrupt or stabilize FtsZ protofilaments. Microscopy strategies are commonly used to characterize FtsZ inhibitors in vivo. Disruption of the Z-ring causes an inhibition of cell division, thus giving the iconic filamentous cell phenotype observed using microscopy.4,125 Further microscopy studies have utilized ΔsulA mutants to determine that the filamentation phenotypes are SulA-independent.88,96,122,184 By use of epifluorescence microscopy techniques, the mislocalization of the Z-ring can be visualized using an anti-FtsZ antibody88,92,106,111,118,122,179 or functional fluorescently tagged FtsZ.99,101,129,184 Additionally, the coupling of a chromosome fluorophore (such as 60) with Z-ring localization experiments establishes whether an inhibitor alters chromosome segregation.88,92,106,111,118,122,129,179 In summary, FtsZ inhibitors are currently characterized in vivo as having a SulA-independent filamentation phenotype with a mislocalized FtsZ ring and no alteration of chromosome segregation. 4.6.1. Challenges Associated with Different Characterization Techniques for FtsZ Inhibitors. A challenge in the search for bacterial cell division inhibitors is differentiating “signal” (i.e., bona fide inhibitors) from “noise”(i.e., nonspecific binders or compounds that target other aspects of the cell and are translated into alterations in FtsZ activity). A variety of different stimuli can trigger FtsZ inhibition, including accumulation of drug aggregates, induction of DNA damage, changes in transmembrane potential, and targeting the activity of proteins positioned upstream of FtsZ, all of which may present the sought-after filamentation phenotype of whole-cell screens. Beyond the PAIN compounds that increasingly turn out “false positives” in bioassays and screening libraries,202,203 druglike molecules can also give spurious results due to nonspecific interactions of drug aggregates with proteins or promiscuous drug−protein interactions.97,204 These aggregates arise from the drug molecules forming organic, often hydrophobic particles in the aqueous environment of a bioassay. 6989 DOI: 10.1021/acs.jmedchem.5b01098 J. Med. Chem. 2016, 59, 6975−6998 Journal of Medicinal Chemistry Perspective development; however many aspects of this regulatory network remain enigmatic. Its central position in cell division highlights FtsZ as a prime candidate for chemotherapeutic strategies, and yet after nearly 2 decades of research on FtsZ inhibitors no inhibitors have emerged for clinical use. The plethora of papers on FtsZ antagonists and lack of multiple, potent inhibitors of FtsZ suggest that finding small molecules to target this protein is challenging. When the activity of these molecules is considered in the context of the many potent inhibitors of tubulin that have been clinically developed, the medicinal chemistry of FtsZ appears to still be stuck in very early stages of development and the field is faced with an important question: Why does this large discovery bandgap exist? One possible explanation is that FtsZ is a much harder protein to drug than tubulin. Comparisons of the crystal structures indicate that tubulin contains numerous regions for binding small molecules, while FtsZ has fewer regions in which molecules can bind, thereby reducing the probability of finding an inhibitor that binds FtsZ antagonistically. Another related explanation is that there is still very little crystal structure data on FtsZ compared to tubulin. Although there are >30 crystal structures deposited in PDB, ∼30% of them are of S. aureus FtsZ and only one has a non-nucleotide small molecule bound in the structure, 42, which provides limited information on how compounds can alter FtsZ structure and activity. 42 binds to S. aureus FtsZ in a flat orientation and appears to affect polymerization by shifting the H7 helix marginally. However, the general lack of crystallographic data makes it difficult to learn how to design better inhibitors using structural biology data. Additional structural biology data of FtsZ bound to the most potent inhibitors could provide insight into design principles; however the lack of available potent FtsZ inhibitors limits this approach. A second possibility centers upon current chemical libraries, which do not often include new molecular entities that contain structural features that are a hallmark of successful clinical antibiotics. Commercial molecular libraries remain locked in a mindset of Lipinski’s rules, which has been successful for identifying inhibitors of individual proteins and compounds active in eukaryotic cells (e.g., mammalian cells) but is not particularly effective for targeting bacteria. Other sets of rules for molecular properties that improve bacterial uptake (e.g., Moser’s rules)212 may be a more helpful tool for curating small molecule libraries for families of molecules that may have antibacterial properties. Specifically, these molecules may have improved transport properties across bacterial membranes and may be poor substrates for transport out of cells through efflux pumps. Limitations of chemical space in synthetic chemical libraries would benefit from following other druglikeness rules for antibiotic drug development. One way to fill this void in the chemical space of commercial libraries is to refocus on natural products and libraries that contain secondary metabolites; here dereplication and metagenomic techniques may be particularly effective. One challenge with FtsZ however remains that it is widely conserved among bacteria, which reduces the possibility that bacteria evolved secondary metabolites to inhibit FtsZ in other cells. For this mechanism to be possible, antibiotic-producing bacteria would require a chaperone that keeps the inhibitor bound until it is secreted. Alternatively, bacteria may evolve mutated forms of FtsZ that have reduced binding to secondary metabolites that they secrete to inhibit the growth of competing bacteria. Consequently, validating that FtsZ inhibitors do not target penicillin-binding proteins or DNA gyrase is an important step in characterizing new drugs.210,211 Surprisingly, some FtsZ inhibitors, such as the zantrins, compounds 37−41, and hemi-10, do not filament cells (or do only very minimally), yet they mislocalize the Z-ring.122 These observations lead to questions surrounding whether FtsZ is the primary target of these compounds. Additionally, the mislocalization of the Z-ring can be attributed to many factors that precede its formation. Positive protein regulators of FtsZ are involved in localizing FtsZ to the center of the cell (Table 2). Consequently, their inhibition could result in a misplacement of the Z-ring. As discussed previously in section 3.3, the localization of cytoskeletal and cell division proteins has been shown to be sensitive to membrane depolarization. The mislocalization of the Z-ring can also be attributed to compromised membranes and a disrupted membrane potential.171 Therefore, the characterization of a possible FtsZ inhibitor should be accompanied by determining whether it affects the membrane. 4.6.2. Strategies To Improve Screening and Characterization Approaches for New FtsZ Inhibitors. To improve the chances of identifying new FtsZ inhibitors, new screening methods should explore new genetic approaches and in vivo techniques. In vivo high-throughput screens with clever cellbased reporter systems provide information about antibacterial potency. Using overexpression strains of FtsZ or an enzyme that interacts with it (Table 2) would distinguish small molecule inhibitors whose antibacterial activity is reversed by overexpression of these enzymes. In accordance with protein− protein interactions, a small molecule activator of SulA (“fake” DNA damage), ClpXP (proteolytic activity), or SlmA (pseudo chromosome mis-segregation) would theoretically cause an indirect stalling of the Z-ring. Many recent detection techniques utilizing new instruments could be implemented for in vivo screens. Microscopes and flow cytometers have been used for high-throughput screening by incorporating stage attachments and detectors that facilitate the use of 96-well plates. Using a 96-well plate microscopy assay, one could observe the cell filamentation and mislocalization of the Z-ring on a large scale that employs more individual compound treatments than are possibly using serial, one-at-atime assays. Flow cytometers can simultaneously measure many aspects of cell physiology (i.e., the length of cells) and the intensity of a chosen fluorescent probe (i.e., 60) in seconds. An experiment could entail grouping all of the in vivo characteristics of a small molecule into one flow cytometry experiment using a ΔsulA lexA (ind−) mutant to control for SOS-dependent cell filamentation, a fluorescently tagged FtsZ to observe Z-ring mislocalization, and a quantifiable DNA stain (such as Picogreen) to quantify the number of chromosomes per nucleoid. The translation of biophysical assays from other areas of biology could be useful for screening small molecule libraries to identify new inhibitors of FtsZ. 5. SUMMARY AND FUTURE DIRECTIONS Bacteria use a variety of regulatory mechanisms to influence the initiation and progression of cell division, many of which ultimately hinge on the essential cell division protein FtsZ. These mechanisms ensure spatiotemporal coordination of cell division and other biological processes necessary for cell viability. Recent studies have uncovered new biochemistry related to cell division that can be exploited for antibiotic 6990 DOI: 10.1021/acs.jmedchem.5b01098 J. Med. Chem. 2016, 59, 6975−6998 Journal of Medicinal Chemistry Perspective Biographies However, the lack of divergence among FtsZ indicates that it may be difficult to mutate the protein and retain its enzymatic function, assembly into filaments, force generation, and contacts with other proteins. These characteristics may have prevented genetic drift of FtsZ and dampened the development of mechanisms of chemical warfare for inhibiting cell division through targeting FtsZ. These considerations reduce the feasibility of secondary metabolites that evolved to target FtsZ. Perhaps some of the exciting new techniques for secondary metabolite identification213 will turn up new compounds with activity against FtsZ and motivate the field to dig deeper into natural products. One of the lessons learned through an analysis of FtsZ inhibitors is the value of in-depth mechanism of action studies to confirm on-target binding and rule out indirect mechanisms of FtsZ antagonism (e.g., triggering the SOS pathway, aggregation and promiscuous binding to proteins, and altering cell membrane potential). Cell filamentation and Z-ring mislocalization are characteristic phenotypes of FtsZ inhibitors; however they can be caused by many different mechanisms. FtsZ studies reveal an aspect of medicinal chemistry that is important for drug design and development: the role of structural biology in confirming and characterizing binding interactions to the target. The lack of small molecules that have been solved in cocrystal structures to FtsZ indicates either a very limited repertoire of current drugs that are bona fide inhibitors of FtsZ or an intrinsic challenge of crystallizing FtsZ and solving the structure with a non-natural compound bound. Targeting cell division with small molecules can leverage many of the different biochemical steps (Table 2) that are connected to FtsZ and cytokinesis. There are a variety of in vivo assays that can be used (or new methods that can be engineered) to qualitatively or quantitatively measure the inhibition of cell division and are compatible with highthroughput screening methods. Bacterial cell division remains an active area of fundamental research, and inhibitors of specific proteins that participate in this process may be important tools for studying the biochemical and biophysical mechanisms that are involved. Although 42 has become the canonical FtsZ inhibitor, it is only effective against S. aureus FtsZ and, for the reasons highlighted earlier, it has provided limited understanding of how to design molecules to target this protein. New families of clinical antibiotics against Gram-negative bacteria that display multidrug resistance214,215 may motivate the discovery of cell division inhibitors against these organisms. FtsZ remains an attractive target for inhibiting division in Gram-negative bacteria. Potent antagonists may have a dual use in understanding how bacteria coordinate the multiple steps of cell division and as antimicrobial agents, which may lead to new clinical antibiotics for chemotherapies. ■ Katherine A. Hurley received her B.S. in Chemistry with a pharmaceutical emphasis at University of CaliforniaDavis, CA, and completed research studies under the supervision of Professor Jared T. Shaw. She is currently pursuing a Ph.D. degree in Pharmaceutical Sciences from the School of Pharmacy at the University of WisconsinMadison, WI, under the supervision of Douglas Weibel. Her present research involves discovering and characterizing new antibiotics as chemotherapeutic agents and chemical biology probes. Thiago M. A. Santos obtained his B.S. in Biological Sciences and a M.S. degree in Agricultural Microbiology from Universidade Federal de Viçosa, Brazil, and pursued predoctoral studies in the College of Veterinary Medicine at Cornell University, NY. He is currently pursuing a Ph.D. degree in the Microbiology Doctoral Training Program at the University of WisconsinMadison, WI, under the supervision of Douglas Weibel. His current research deciphers the molecular mechanisms of protein localization in bacteria and the mode of action of novel antimicrobial drugs. Gabriella M. Nepomuceno received her B.S. in Chemistry at University of CaliforniaSanta Cruz, CA, and completed research studies under the supervision of Professor Bakthan Singaram. She received a Ph.D. degree in Chemistry from the University of CaliforniaDavis, CA, under the supervision of Jared T. Shaw. Her research involved designing and synthesizing molecular probes in chemical biology and organic methodology. Valerie Huynh received her B.S. in Pharmaceutical Chemistry from the University of CaliforniaDavis, CA, under the supervision of Jared T. Shaw. She received a M.S. degree in Pharmaceutical Chemistry at the same institution. Her research involved synthesizing molecular probes for medicinal chemistry. Currently, she is a Research Associate at Gilead Sciences, Inc. Jared T. Shaw received his Ph.D. in Chemistry from Keith Woerpel at University of CaliforniaIrvine and then moved to Harvard University, MA, as an NIH Postdoctoral Fellow with David Evans. He became an Institute Fellow at the Institute for Chemistry and Cell Biology (ICCB) at Harvard Medical School where he helped found the Center for Chemical Methodology and Library Development (CMLD), which later became part of the Broad Institute of Harvard and Massachusetts Institute of Technology. He is currently an Associate Professor of Chemistry at the University of California Davis, CA, and he currently works on the development of new methods for the synthesis of natural products and other complex molecules that modulate biological phenomena. Douglas B. Weibel received his B.S. degree in Chemistry from the University of Utah, was a Fulbright Fellow at Tohoku University, Japan (with Yoshinori Yamamoto), and received his Ph.D. in Chemistry from Cornell University, NY (with Jerrold Meinwald). He was a Postdoctoral Fellow at Harvard University, MA (with George Whitesides). He is currently an Associate Professor of Biochemistry, Chemistry, and Biomedical Engineering at the University of WisconsinMadison, WI, and his research spans the fields of biochemistry, biophysics, chemistry, materials science and engineering, and microbiology. AUTHOR INFORMATION Corresponding Authors *J.T.S.: e-mail, [email protected]; phone, +1 (530) 752-9979. *D.B.W.: e-mail, [email protected]; phone, +1 (608) 890-1342; fax, +1 (608) 265-0764. ■ ACKNOWLEDGMENTS Due to space constraints, we were unable to cite all of the research on FtsZ; any occlusions were unintentional. Research on antibiotics in the Weibel laboratory has been supported by the Human Frontiers Science Program (Grant RGY0076/2013), the NIH (Grant 1DP2OD008735), the Wisconsin Alumni Research Author Contributions # K.A.H. and T.M.A.S. contributed equally to this article. Notes The authors declare no competing financial interest. 6991 DOI: 10.1021/acs.jmedchem.5b01098 J. Med. Chem. 2016, 59, 6975−6998 Journal of Medicinal Chemistry Perspective (19) Vollmer, W. The Prokaryotic Cytoskeleton: A Putative Target for Inhibitors and Antibiotics? Appl. Microbiol. Biotechnol. 2006, 73, 37−47. (20) Addinall, S. G.; Holland, B. The Tubulin Ancester, FtsZ, Draughtsman, Designer and Driving Force for Bacterial Cytokinesis. J. Mol. Biol. 2002, 318, 219−236. (21) Li, X.; Ma, S. Advances in the Discovery of Novel Antimicrobials Targeting the Assembly of Bacterial Cell Division Protein FtsZ. Eur. J. Med. Chem. 2015, 95, 1−15. (22) Hong, W.; Xie, J. Progress of FtsZ Inhibitors as Novel Antibiotics Leads. Crit. Rev. Eukaryotic Gene Expression 2013, 23, 327− 338. (23) Ma, S.; Ma, S. The Development of FtsZ Inhibitors as Potential Antibacterial Agents. ChemMedChem 2012, 7, 1161−1172. (24) Schaffner-Barbero, C.; Martín-Fontecha, M.; Chacón, P.; Andreu, J. M. Targeting the Assembly of Bacterial Cell Division Protein FtsZ. with Small Molecules. ACS Chem. Biol. 2012, 7, 269− 277. (25) Awasthi, D.; Kumar, K.; Ojima, I. Therapeutic Potential of FtsZ Inhibition: A Patent Perspective. Expert Opin. Ther. Pat. 2011, 21, 657−679. (26) Huang, Q.; Tonge, P. J.; Slayden, R. A.; Kirikae, T.; Ojima, I. 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A.; Chacon, P.; Andreu, J. M. Tubulin Secondary Structure Analysis, Limited Proteolysis Sites, and Homology to FtsZ. Biochemistry 1996, 35, 14203−14215. (33) Shen, B.; Lutkenhaus, J. The Conserved C-Terminal Tail of FtsZ Is Required for the Septal Localization and Division Inhibitory Activity of MinC/MinD. Mol. Microbiol. 2009, 72, 410−424. (34) Ma, X.; Margolin, W. Genetic and Functional Analyses of the Conserved C-Terminal Core Domain of Escherichia coli FtsZ. J. Bacteriol. 1999, 181, 7531−7544. (35) Haney, S. A.; Glasfeld, E.; Hale, C.; Keeney, D.; He, Z.; de Boer, P. A. J. Genetic Analysis of the Escherichia coli FtsZ·ZipA Interaction in the Yeast Two-Hybrid System. J. Biol. Chem. 2001, 276, 11980−11987. (36) Hale, C. A.; Rhee, A. C.; de Boer, P. A. J. ZipA-Induced Bundling of FtsZ Polymers Mediated by an Interaction between CTerminal Domains. J. Bacteriol. 2000, 182, 5153−5166. (37) Singh, J. K.; Makde, R. D.; Kumar, V.; Panda, D. A Membrane Protein, EzrA, Regulates Assembly Dynamics of FtsZ by Interacting with the C-Terminal Tail of FtsZ. Biochemistry 2007, 46, 11013− 11022. (38) Camberg, J. L.; Hoskins, J. R.; Wickner, S. ClpXP Protease Degrades the Cytoskeletal Protein, FtsZ, and Modulates FtsZ Polymer Dynamics. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 10614−10619. (39) Singh, J. K.; Makde, R. D.; Kumar, V.; Panda, D. SepF Increases the Assembly and Bundling of FtsZ Polymers and Stabilizes FtsZ Protofilaments by Binding along Its Length. J. Biol. Chem. 2008, 283, 31116−31124. (40) Buske, P. J.; Levin, P. A. Extreme C Terminus of Bacterial Cytoskeletal Protein FtsZ Plays Fundamental Role in Assembly Independent of Modulatory Proteins. J. Biol. Chem. 2012, 287, 10945− 10957. Foundation, and the USDA (Grant WIS01594). Research on inhibitors of cell division in the Shaw laboratory is supported by NIH/NIAID (Grants R01A108093, R01A08093-04S1). ■ ABBREVIATIONS USED ANS, 8-anilinonaphthalene-1-sulfonic acid; ADME, absorption, distribution, metabolism, and excretion; CCCP, carbonyl cyanide m-chlorophenyl hydrazone; DAPI, 4′,6-diamidino-2phenylindole; EGS, external guide sequence; FDA, Food and Drug Administration; FRAP, fluorescence recovery after photobleaching; FRET, fluorescence resonance energy transfer; GDP, guanosine diphosphate; GTP, guanosine triphosphate; pN, piconewton; OTBA, 3-{5-[4-oxo-2-thioxo-3-(3trifluoromethylphenyl)thiazolidin-5-ylidenemethyl]furan-2-yl}benzoic acid; PAIN, pan-assay interference; ROCS, rapid overlay of chemical structures; SAR, structure−activity relationship; UV, ultraviolet ■ REFERENCES (1) Walsh, C. T. Validated Targets and Major Antibiotic Classes. In Antibiotics: Actions, Origins, Resistance; Walsh, C. T., Ed.; ASM Press: Washington, DC, 2003; pp 11−79. 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