Targeting the Bacterial Division Protein FtsZ

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*,‡,∥,⊥

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†

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

ABSTRACT: Similar to its eukaryotic counterpart, the prokaryotic cytoskeleton is essential for the structural and mechanical properties of bacterial cells. The essential protein FtsZ is a central player in the cytoskeletal family, forms a cytokinetic ring at mid-cell, and recruits the division machinery to orchestrate cell division. Cells depleted of or lacking functional FtsZ do not divide and grow into long filaments that eventually lyse. FtsZ has been studied extensively as a target for antibacterial development. In this Perspective, we review the structural and biochemical properties of FtsZ, its role in cell biochemistry and physiology, the different mechanisms of inhibiting FtsZ, small molecule antagonists (including some misconceptions about mechanisms of action), and their discovery strategies. This collective information will inform chemists on different aspects of FtsZ that can be (and have been) used to develop successful strategies for devising new families of cell division inhibitors.

1. INTRODUCTION: TARGETING THE BACTERIAL PROTEIN FtsZ An increase of multidrug resistance to antibiotics among pathogenic strains of bacteria and the lack of innovation in the discovery of new antibacterial agents punctuate the need for new chemotherapeutic strategies. One approach to new strategies is the identification, characterization, and exploration of new molecular targets for antibiotic development, which is currently in vogue. Historically, all known clinical antibiotics target one of the following bacterial structures and cellular processes: (1) DNA replication; (2) transcription; (3) translation; (4) peptidoglycan biosynthesis; (5) folate biosynthesis; (6) the cytoplasmic membrane.1,2 An important, unanswered question is whether additional classes of mechanisms and targets exist for developing new families of antibiotics. The bacterial cytoskeleton is one such family of targets for which clinical antibiotics have not yet emerged. The cytoskeleton is an ancient cellular invention that probably precedes the divergence between eukaryotes and prokaryotes.3 The bacterial cytoskeleton consists of families of proteins essential for the physiological and structural properties of cells, including cell division,4,5 cell wall growth,6,7 cell shape determination/ maintenance,8,9 DNA segregation,10 and protein localization10 (Table 1). Because its integrity is important to cell viability, the bacterial cytoskeleton has been a topic of discussion for the development of antibacterial compounds over the past 2 decades. © 2016 American Chemical Society

The essential cytoskeletal cell division protein FtsZ (named after the filamenting temperature-sensitive mutant Z) is an essential GTPase structurally related to eukaryotic tubulins11−13 and highly conserved in bacteria and archaea.14,15 During cell division, FtsZ forms a ringlike structure at the site of division and functions as a scaffold for the assembly of a multiprotein complex (referred to as the “divisome”) essential for cell viability. Not surprisingly, FtsZ, as well as proteins that interact directly with and regulate the activity of FtsZ, has emerged as a prime target for antibacterial development.16 The use of FtsZ as an antibacterial drug target has been reviewed,17,18 and its structural biology16,19,20 and inhibition with small molecules have been discussed.21−25 Specifically, targeting FtsZ with small molecules as a defense against tuberculosis has also been extensively reviewed.26−28 In this review, we explore the latest developments of classes of small molecules and inhibitors targeting FtsZ and evaluate the challenges and future directions of this field of antibiotic research.

2. STRUCTURE AND FUNCTION OF FtsZ 2.1. FtsZ Structure. FtsZ shares 40−50% sequence identity across most bacterial and archaeal species and has a threedimensional structure that is similar to the structure of α- and Received: July 14, 2015 Published: January 12, 2016 6975

DOI: 10.1021/acs.jmedchem.5b01098 J. Med. Chem. 2016, 59, 6975−6998

Journal of Medicinal Chemistry

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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 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



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A drug whose dose−response curve deviates from the standard sigmoidal shape and instead forms a bell-shaped curve at higher concentrations of drug should be scrutinized as a potential aggregating molecule.205 One method for detecting the presence of drug aggregates is implementing a detergentbased control using Triton X-100, which dissolves drug−drug aggregates.206 However, Triton X-100 has been shown to disrupt the protein−protein interactions between the tubulin polymers as evidenced by electron microscopy.121 Nevertheless, a cross-comparison of Triton X-100 versus centrifugation or increasing protein concentration showed similar results for 37, 42, and 20.97 Furthermore, Triton X-100 has been used in control experiments with derivatives of 24113 and analogs of 2.91 Bona fide inhibitors of FtsZ will produce the same dose− response curve with or without an aggregation test, while an aggregator will result in a loss of activity at any drug concentration. In vitro measurements of GTP hydrolysis by FtsZ are complicated by the complexity of the biological coupling between GTPase activity and FtsZ protofilament formation. A thorough reexamination of several reported FtsZ inhibitors indicates two are aggregators (e.g., 7 and 37); one is a PAINs compound (e.g., 32), and several inhibitors have activity that vary significantly from earlier reports (e.g., 9).97 Aggregation is a problem for in vitro assays, such as GTPase activity measurements and light scattering experiments, in which a nonspecific aggregation effect of small molecule inhibitors with FtsZ monomers impedes polymerization. FtsZ protofilaments and bundles can be observed by electron microscopy; however this technique is unable to decouple whether a decreasing FtsZ polymerization rate and the change in the number of protofilaments are a result of aggregation or an inhibitor binding to FtsZ specifically. FRET assays can be used to transcend this limitation. One population of FtsZ proteins can be labeled with a donor fluorophore (a FRET donor) and another with an acceptor fluorophore (a FRET acceptor). Polymerization of FtsZ monomers creates a spatial distribution of fluorophores; a donor and acceptor positioned adjacent to each other will produce a FRET signal.207 An inhibitor that disrupts FtsZ polymerization may reduce the FRET signal. Cell filamentation arises in response to alterations in the topological state or structure of DNA. As mentioned in section 3.2.3, when the SOS response is triggered in response to DNA damage, SulA inhibits FtsZ polymerization (Table 2) and cell filamentation occurs. However, bacteria can filament via SulA-independent mechanisms after the SOS response is triggered by DNA damage, perturbation of DNA topology, and stalling of the DNA replication fork.185,208 The first step in the SOS response is controlled by the RecA-activated selfcleavage of LexA, a transcriptional repressor of all the SOS response genes.208 Therefore, the use of a ΔsulA mutant with a noncleavable LexA repressor (lexA (ind−)) is more informative than a ΔsulA mutant, as the activation of all of the SOS response genes and their contribution to SulA-independent filamentation should be assessed. As referred above in section 2.2, the depletion of other Fts proteins yields filamentous cells that are multinucleate (i.e., they have multiple copies of chromosomes that are evenly spaced along the length of the filament),209 suggesting that inhibiting Fts proteins (other than FtsZ) produces a filamentous phenotype. Inhibiting some clinically relevant antibacterial targets (such as inhibitors of peptidoglycan biosynthesis and DNA supercoiling) produces a filamentous cell phenotype.

The in vitro measurement of GTP hydrolysis activity has become a standard assay for FtsZ inhibitors. As mentioned earlier, target-based high-throughput screens were designed to monitor GTPase activity by measuring the release of inorganic phosphate using a malachite green dye assay99,106,111,118,135,137 or coupled enzyme assays.122,200 A common secondary assay to evaluate compounds in vitro is monitoring the inhibition or stabilization of FtsZ protofilaments and bundles using light scattering or electron microscopy.88,89,101,105,106,115,118,127,135,137,179,201 Further in vitro studies can be performed to characterize FtsZ inhibition by evaluating the intrinsic properties of FtsZ. The conformational changes of FtsZ and its bundles in the presence of inhibitor are measurable by far-UV circular dichroism.100,105,106,118 Many assays have been developed to demonstrate compounds binding to FtsZ directly. A simple method is to perform a sedimentation assay that takes advantage of the formation of insoluble protofilaments as FtsZ polymerizes, which precipitate out of solution and can be collected by centrifugation. If an inhibitor prevents polymerization, the amount of sedimented FtsZ decreases; conversely, the amount of sedimented FtsZ increases if the inhibitor stabilizes protofilaments.111,118,122,127,135,179 Several examples of competitive binding assays have been reported that use fluorescent inhibitors,106,108 fluorescent probes,111 fluorescent GTP nonhydrolyzable analogs,99,111,179 and modification of FtsZ with fluorescein96 or tryptophan residues.92,118,179 All of these in vitro techniques facilitate characterizing compounds as GTPase inhibitors or activators that disrupt or stabilize FtsZ protofilaments. Microscopy strategies are commonly used to characterize FtsZ inhibitors in vivo. Disruption of the Z-ring causes an inhibition of cell division, thus giving the iconic filamentous cell phenotype observed using microscopy.4,125 Further microscopy studies have utilized ΔsulA mutants to determine that the filamentation phenotypes are SulA-independent.88,96,122,184 By use of epifluorescence microscopy techniques, the mislocalization of the Z-ring can be visualized using an anti-FtsZ antibody88,92,106,111,118,122,179 or functional fluorescently tagged FtsZ.99,101,129,184 Additionally, the coupling of a chromosome fluorophore (such as 60) with Z-ring localization experiments establishes whether an inhibitor alters chromosome segregation.88,92,106,111,118,122,129,179 In summary, FtsZ inhibitors are currently characterized in vivo as having a SulA-independent filamentation phenotype with a mislocalized FtsZ ring and no alteration of chromosome segregation. 4.6.1. Challenges Associated with Different Characterization Techniques for FtsZ Inhibitors. A challenge in the search for bacterial cell division inhibitors is differentiating “signal” (i.e., bona fide inhibitors) from “noise”(i.e., nonspecific binders or compounds that target other aspects of the cell and are translated into alterations in FtsZ activity). A variety of different stimuli can trigger FtsZ inhibition, including accumulation of drug aggregates, induction of DNA damage, changes in transmembrane potential, and targeting the activity of proteins positioned upstream of FtsZ, all of which may present the sought-after filamentation phenotype of whole-cell screens. Beyond the PAIN compounds that increasingly turn out “false positives” in bioassays and screening libraries,202,203 druglike molecules can also give spurious results due to nonspecific interactions of drug aggregates with proteins or promiscuous drug−protein interactions.97,204 These aggregates arise from the drug molecules forming organic, often hydrophobic particles in the aqueous environment of a bioassay. 6989



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development; however many aspects of this regulatory network remain enigmatic. Its central position in cell division highlights FtsZ as a prime candidate for chemotherapeutic strategies, and yet after nearly 2 decades of research on FtsZ inhibitors no inhibitors have emerged for clinical use. The plethora of papers on FtsZ antagonists and lack of multiple, potent inhibitors of FtsZ suggest that finding small molecules to target this protein is challenging. When the activity of these molecules is considered in the context of the many potent inhibitors of tubulin that have been clinically developed, the medicinal chemistry of FtsZ appears to still be stuck in very early stages of development and the field is faced with an important question: Why does this large discovery bandgap exist? One possible explanation is that FtsZ is a much harder protein to drug than tubulin. Comparisons of the crystal structures indicate that tubulin contains numerous regions for binding small molecules, while FtsZ has fewer regions in which molecules can bind, thereby reducing the probability of finding an inhibitor that binds FtsZ antagonistically. Another related explanation is that there is still very little crystal structure data on FtsZ compared to tubulin. Although there are >30 crystal structures deposited in PDB, ∼30% of them are of S. aureus FtsZ and only one has a non-nucleotide small molecule bound in the structure, 42, which provides limited information on how compounds can alter FtsZ structure and activity. 42 binds to S. aureus FtsZ in a flat orientation and appears to affect polymerization by shifting the H7 helix marginally. However, the general lack of crystallographic data makes it difficult to learn how to design better inhibitors using structural biology data. Additional structural biology data of FtsZ bound to the most potent inhibitors could provide insight into design principles; however the lack of available potent FtsZ inhibitors limits this approach. A second possibility centers upon current chemical libraries, which do not often include new molecular entities that contain structural features that are a hallmark of successful clinical antibiotics. Commercial molecular libraries remain locked in a mindset of Lipinski’s rules, which has been successful for identifying inhibitors of individual proteins and compounds active in eukaryotic cells (e.g., mammalian cells) but is not particularly effective for targeting bacteria. Other sets of rules for molecular properties that improve bacterial uptake (e.g., Moser’s rules)212 may be a more helpful tool for curating small molecule libraries for families of molecules that may have antibacterial properties. Specifically, these molecules may have improved transport properties across bacterial membranes and may be poor substrates for transport out of cells through efflux pumps. Limitations of chemical space in synthetic chemical libraries would benefit from following other druglikeness rules for antibiotic drug development. One way to fill this void in the chemical space of commercial libraries is to refocus on natural products and libraries that contain secondary metabolites; here dereplication and metagenomic techniques may be particularly effective. One challenge with FtsZ however remains that it is widely conserved among bacteria, which reduces the possibility that bacteria evolved secondary metabolites to inhibit FtsZ in other cells. For this mechanism to be possible, antibiotic-producing bacteria would require a chaperone that keeps the inhibitor bound until it is secreted. Alternatively, bacteria may evolve mutated forms of FtsZ that have reduced binding to secondary metabolites that they secrete to inhibit the growth of competing bacteria.

Consequently, validating that FtsZ inhibitors do not target penicillin-binding proteins or DNA gyrase is an important step in characterizing new drugs.210,211 Surprisingly, some FtsZ inhibitors, such as the zantrins, compounds 37−41, and hemi-10, do not filament cells (or do only very minimally), yet they mislocalize the Z-ring.122 These observations lead to questions surrounding whether FtsZ is the primary target of these compounds. Additionally, the mislocalization of the Z-ring can be attributed to many factors that precede its formation. Positive protein regulators of FtsZ are involved in localizing FtsZ to the center of the cell (Table 2). Consequently, their inhibition could result in a misplacement of the Z-ring. As discussed previously in section 3.3, the localization of cytoskeletal and cell division proteins has been shown to be sensitive to membrane depolarization. The mislocalization of the Z-ring can also be attributed to compromised membranes and a disrupted membrane potential.171 Therefore, the characterization of a possible FtsZ inhibitor should be accompanied by determining whether it affects the membrane. 4.6.2. Strategies To Improve Screening and Characterization Approaches for New FtsZ Inhibitors. To improve the chances of identifying new FtsZ inhibitors, new screening methods should explore new genetic approaches and in vivo techniques. In vivo high-throughput screens with clever cellbased reporter systems provide information about antibacterial potency. Using overexpression strains of FtsZ or an enzyme that interacts with it (Table 2) would distinguish small molecule inhibitors whose antibacterial activity is reversed by overexpression of these enzymes. In accordance with protein− protein interactions, a small molecule activator of SulA (“fake” DNA damage), ClpXP (proteolytic activity), or SlmA (pseudo chromosome mis-segregation) would theoretically cause an indirect stalling of the Z-ring. Many recent detection techniques utilizing new instruments could be implemented for in vivo screens. Microscopes and flow cytometers have been used for high-throughput screening by incorporating stage attachments and detectors that facilitate the use of 96-well plates. Using a 96-well plate microscopy assay, one could observe the cell filamentation and mislocalization of the Z-ring on a large scale that employs more individual compound treatments than are possibly using serial, one-at-atime assays. Flow cytometers can simultaneously measure many aspects of cell physiology (i.e., the length of cells) and the intensity of a chosen fluorescent probe (i.e., 60) in seconds. An experiment could entail grouping all of the in vivo characteristics of a small molecule into one flow cytometry experiment using a ΔsulA lexA (ind−) mutant to control for SOS-dependent cell filamentation, a fluorescently tagged FtsZ to observe Z-ring mislocalization, and a quantifiable DNA stain (such as Picogreen) to quantify the number of chromosomes per nucleoid. The translation of biophysical assays from other areas of biology could be useful for screening small molecule libraries to identify new inhibitors of FtsZ.

5. SUMMARY AND FUTURE DIRECTIONS Bacteria use a variety of regulatory mechanisms to influence the initiation and progression of cell division, many of which ultimately hinge on the essential cell division protein FtsZ. These mechanisms ensure spatiotemporal coordination of cell division and other biological processes necessary for cell viability. Recent studies have uncovered new biochemistry related to cell division that can be exploited for antibiotic 6990



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Biographies

However, the lack of divergence among FtsZ indicates that it may be difficult to mutate the protein and retain its enzymatic function, assembly into filaments, force generation, and contacts with other proteins. These characteristics may have prevented genetic drift of FtsZ and dampened the development of mechanisms of chemical warfare for inhibiting cell division through targeting FtsZ. These considerations reduce the feasibility of secondary metabolites that evolved to target FtsZ. Perhaps some of the exciting new techniques for secondary metabolite identification213 will turn up new compounds with activity against FtsZ and motivate the field to dig deeper into natural products. One of the lessons learned through an analysis of FtsZ inhibitors is the value of in-depth mechanism of action studies to confirm on-target binding and rule out indirect mechanisms of FtsZ antagonism (e.g., triggering the SOS pathway, aggregation and promiscuous binding to proteins, and altering cell membrane potential). Cell filamentation and Z-ring mislocalization are characteristic phenotypes of FtsZ inhibitors; however they can be caused by many different mechanisms. FtsZ studies reveal an aspect of medicinal chemistry that is important for drug design and development: the role of structural biology in confirming and characterizing binding interactions to the target. The lack of small molecules that have been solved in cocrystal structures to FtsZ indicates either a very limited repertoire of current drugs that are bona fide inhibitors of FtsZ or an intrinsic challenge of crystallizing FtsZ and solving the structure with a non-natural compound bound. Targeting cell division with small molecules can leverage many of the different biochemical steps (Table 2) that are connected to FtsZ and cytokinesis. There are a variety of in vivo assays that can be used (or new methods that can be engineered) to qualitatively or quantitatively measure the inhibition of cell division and are compatible with highthroughput screening methods. Bacterial cell division remains an active area of fundamental research, and inhibitors of specific proteins that participate in this process may be important tools for studying the biochemical and biophysical mechanisms that are involved. Although 42 has become the canonical FtsZ inhibitor, it is only effective against S. aureus FtsZ and, for the reasons highlighted earlier, it has provided limited understanding of how to design molecules to target this protein. New families of clinical antibiotics against Gram-negative bacteria that display multidrug resistance214,215 may motivate the discovery of cell division inhibitors against these organisms. FtsZ remains an attractive target for inhibiting division in Gram-negative bacteria. Potent antagonists may have a dual use in understanding how bacteria coordinate the multiple steps of cell division and as antimicrobial agents, which may lead to new clinical antibiotics for chemotherapies.

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Katherine A. Hurley received her B.S. in Chemistry with a pharmaceutical emphasis at University of CaliforniaDavis, CA, and completed research studies under the supervision of Professor Jared T. Shaw. She is currently pursuing a Ph.D. degree in Pharmaceutical Sciences from the School of Pharmacy at the University of WisconsinMadison, WI, under the supervision of Douglas Weibel. Her present research involves discovering and characterizing new antibiotics as chemotherapeutic agents and chemical biology probes. Thiago M. A. Santos obtained his B.S. in Biological Sciences and a M.S. degree in Agricultural Microbiology from Universidade Federal de Viçosa, Brazil, and pursued predoctoral studies in the College of Veterinary Medicine at Cornell University, NY. He is currently pursuing a Ph.D. degree in the Microbiology Doctoral Training Program at the University of WisconsinMadison, WI, under the supervision of Douglas Weibel. His current research deciphers the molecular mechanisms of protein localization in bacteria and the mode of action of novel antimicrobial drugs. Gabriella M. Nepomuceno received her B.S. in Chemistry at University of CaliforniaSanta Cruz, CA, and completed research studies under the supervision of Professor Bakthan Singaram. She received a Ph.D. degree in Chemistry from the University of CaliforniaDavis, CA, under the supervision of Jared T. Shaw. Her research involved designing and synthesizing molecular probes in chemical biology and organic methodology. Valerie Huynh received her B.S. in Pharmaceutical Chemistry from the University of CaliforniaDavis, CA, under the supervision of Jared T. Shaw. She received a M.S. degree in Pharmaceutical Chemistry at the same institution. Her research involved synthesizing molecular probes for medicinal chemistry. Currently, she is a Research Associate at Gilead Sciences, Inc. Jared T. Shaw received his Ph.D. in Chemistry from Keith Woerpel at University of CaliforniaIrvine and then moved to Harvard University, MA, as an NIH Postdoctoral Fellow with David Evans. He became an Institute Fellow at the Institute for Chemistry and Cell Biology (ICCB) at Harvard Medical School where he helped found the Center for Chemical Methodology and Library Development (CMLD), which later became part of the Broad Institute of Harvard and Massachusetts Institute of Technology. He is currently an Associate Professor of Chemistry at the University of California Davis, CA, and he currently works on the development of new methods for the synthesis of natural products and other complex molecules that modulate biological phenomena. Douglas B. Weibel received his B.S. degree in Chemistry from the University of Utah, was a Fulbright Fellow at Tohoku University, Japan (with Yoshinori Yamamoto), and received his Ph.D. in Chemistry from Cornell University, NY (with Jerrold Meinwald). He was a Postdoctoral Fellow at Harvard University, MA (with George Whitesides). He is currently an Associate Professor of Biochemistry, Chemistry, and Biomedical Engineering at the University of WisconsinMadison, WI, and his research spans the fields of biochemistry, biophysics, chemistry, materials science and engineering, and microbiology.

AUTHOR INFORMATION

Corresponding Authors

*J.T.S.: e-mail, [email protected]; phone, +1 (530) 752-9979. *D.B.W.: e-mail, [email protected]; phone, +1 (608) 890-1342; fax, +1 (608) 265-0764.

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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



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

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

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