European Journal of Medicinal Chemistry 95 (2015) 1e15

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Mini-Review

Advances in the discovery of novel antimicrobials targeting the assembly of bacterial cell division protein FtsZ Xin Li, Shutao Ma* Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, 44 West Culture Road, Jinan 250012, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 January 2015 Received in revised form 10 March 2015 Accepted 12 March 2015 Available online 14 March 2015

Currently, wide-spread antimicrobials resistance among bacterial pathogens continues being a dramatically increasing and serious threat to public health, and thus there is a pressing need to develop new antimicrobials to keep pace with the bacterial resistance. Filamentous temperature-sensitive protein Z (FtsZ), a prokaryotic cytoskeleton protein, plays an important role in bacterial cell division. It as a very new and promising target, garners special attention in the antibacterial research in the recent years. This review describes not only the function and dynamic behaviors of FtsZ, but also the known natural and synthetic inhibitors of FtsZ. In particular, the small molecules recently developed and the future directions of ideal candidates are highlighted. © 2015 Published by Elsevier Masson SAS.

Keywords: Antimicrobials Biological activity Bacterial cell division Bacterial resistance FtsZ Structure-activity relationship

1. Introduction Bacterial infections remain one of the major public health problems throughout the world in hospital and community settings [1]. In large part, this is attributed to the increasing prevalence of resistant pathogens such as methicillin resistant Staphylococcus aureus (MRSA), vancomycin resistant enterococci (VRE), “superbugs” with New Delhi metallo-b-lactamase-1 (NDM-1) and others due to use/overuse/misuse of antibiotic therapeutics [2]. The resistance of bacteria, making many originally powerful antibiotics in the clinic show either weak or no activity, is developed by undergoing mutations, using efflux mechanisms and applying enzymatic clearance of drugs [3]. In order to counter the attack of the adaptive genetic machinery of bacteria, there is an urgent need for the discovery of new antibacterial agents with full activity against multi-resistant pathogens. Since the efficacious modification of the existing antimicrobials is becoming growingly difficult, targeting novel, unexplored essential proteins to develop new classes of antimicrobials has been considered to be more and more critical for the discovery of noncross resistant antibacterial agents [4]. Filamentous temperature-

sensitive protein Z (FtsZ), as an essential cell-division protein conserved in virtually all eubacteria, archeae and chloroplasts [5], has been validated as a highly promising target for antibacterial intervention [6]. It forms a highly dynamic Z-ring at the center of the cell and then recruits other cell division proteins. After the completion of recruitment, the Z ring contracts, leading to the closure of the septum along with the formation of two daughter cells [7]. Owing to its important role in bacterial cell division and largely unexploited as a new target for antibacterial agents, FtsZ has aroused a great number of interests among many researchers involved in developing new generation of novel antibacterial agents in recent years [8e11]. In this review, we describe not only the function and dynamic behaviors of FtsZ, but also the known natural and synthetic inhibitors of FtsZ, with focus on their mechanism of action, biological activity, availability, and structure-activity relationship (SAR) information. In particular, the small molecules recently developed and the inspirations from their structures as well as the future direction of ideal candidates are highlighted. 2. Structure and function of FTSZ 2.1. FtsZ structure

* Corresponding author. E-mail address: [email protected] (S. Ma). http://dx.doi.org/10.1016/j.ejmech.2015.03.026 0223-5234/© 2015 Published by Elsevier Masson SAS.

FtsZ is composed of an N-terminal enzymatic domain and a long

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X. Li, S. Ma / European Journal of Medicinal Chemistry 95 (2015) 1e15

C-terminal domain with different extensions [12e14]. The enzymatic domain consists of two globular sub-domains, designated as N-terminal subdomain and C-terminal subdomain, which are parted by the central core H7 helix. The N-terminal enzymatic domain contains a nucleotide-binding pocket, and the C-terminal subdomain includes a GTPase-activating pool. At the tail of the long C-terminal extension, there are some functional sites that can recognize the accessory proteins [15,16]. Additionally, the N-terminal enzymatic domain and the long C-terminal domain are linked together through a H5 helix [13]. FtsZ is regarded as the homologue of the eukaryotic cytoskeleton protein tubulin, which may be due to their similarity in structural and functional features. Structurally, FtsZ shares a short fragment of amino acid motif, GGGTGTG, which is nearly identical to the signature sequence, (G/A)GGTGSG, existing in all a, b and g subunit types of tubulin [12,17,18]. Together, FtsZ and tubulin have a common fold, consisting of two domains linked by an a-helix [13]. Conserved residues of FtsZ and tubulin are located in the nucleotide-binding region which is related to protofilament formation [19]. Functionally, similar to tubulin, FtsZ has the polymerization behavior, the potential to bind GTP, and the selfactivated GTPase activity with the active site formed by the interaction of two adjacent monomers [20]. Furthermore, just as the microtubule assembly is influenced by microtubule associated proteins (MAPs), the stability of FtsZ is controlled in vivo by the proteins such as FtsA, ZipA and ZapA [21e24]. While tubulin and FtsZ possess some common features, the two proteins have limited sequence similarity (10
18%) at the protein level [25], and they demonstrate significant differences in the binding mode with nucleotides [26], in the C-terminus amino acid sequence and in the content of a-helices [27]. Moreover, tubulin consists of a and b heterodimers with distinct polarity, whereas FtsZ polymers include only one form of FtsZ monomers [28]. Furthermore, FtsZ protofilaments are associated with each other laterally, different from the longitudinal association in tubulin subunits, leading to the formation of an arc-shaped dimer [29]. These facts provide the opportunity to discover antibacterial agents targeting FtsZ specifically with limited cytotoxicity to eukaryotic cells. 2.2. FtsZ function 2.2.1. Biological role of FtsZ in bacterial cytokinesis FtsZ is a bacteria cytoskeleton protein, playing a critical role in the bacteria cell devision. The “fts” genes products were considered to be related to the cell septum formation [30,31], and then the product FtsZ was discovered to be involved in the intracellular Z ring formation at the mid-cell for the initiation of cell division in 1991 [31]. In the process of cell devision, FtsZ firstly initiates polymerization in the presence of GTP at a single site of the inner membrane in the middle region of the cell. The resulting polymer appears to grow bi-directionally, assembling into a highly dynamic helical structure known as Z-ring at the center of the cell [20,29,32e34]. Subsequently, twelve other downstream proteins at least are recruited to the Z-ring structure, leading to the formation of the divisome complex. Then, the divisome contracts, which contributes to the septum formation and then the final cell division [35]. In addition, the polymerization of FtsZ is precisely regulated by several other proteins [32]. The inhibition of FtsZ can cause the complete absence of septum in the cell division, further resulting in the cell death. Accordingly, FtsZ as a functional protein has the potential to be a novel drug target for the exploration of drugresistance microbial inhibitors.

2.2.2. Dynamics of FtsZ: FtsZ polymerization and GTP hydrolysis FtsZ exists in the cytoplasmic pool in the form of monomers and oligomers. It is reported that there are about 15,000 FtsZ molecules in an Enterococcus coli cell [36] and their concentration remains constant during the entire cell cycle [22]. Of these FtsZ molecules, only about 30% of the molecules participate in Z-ring formation at a given time and the participated FtsZ molecules in Z-ring undergoes rapid exchange with the FtsZ molecules in the cytoplasm with a half-life of nearly eight seconds [37e39]. So, the resulting Z-ring keeps constant balance between assembly and disassembly, and appears to be static in the overall morphology [40]. The assembly of FtsZ does not initiate until the DNA replication is accomplished and the nucleoids are to be segregated [22,41]. Furthermore, the presence of GTP, exceeding a critical concentration of 1e0.5 mM [42e44], is necessary for this process. After the realization of the external conditions, the FtsZ monomers binding GTP assemble cooperatively in a head-to-tail manner to form single-stranded straight protein filaments with a width of 4e5 nm, which are called protofilaments [45,46]. This assembly results in the binding GTP sandwiched between two adjacent FtsZ monomers [43,47,48] and triggers the GTPase activity of FtsZ [49] because the catalytic site is formed with the accomplishment of the assembly at the interface of the two adjacent FtsZ molecules [49]. Then, the protofilaments interact laterally and coalesce into pairs, bundles, sheets, and other condensates [50e52], leading to the formation of the highly dynamic Z-ring structure [43]. In the straight FtsZ polymer, the T7 loop in the ‘upper’ FtsZ monomer is pushed into the nucleotide-binding region of the ‘lower’ FtsZ monomer [53] and the catalytic residues located in the T7 loop are placed near the gphosphate of GTP, resulting in the hydrolysis of the GTP. As the GTP is hydrolyzed to guanosine diphosphate (GDP), the protofilaments switch from the straight to the curved conformation [54]. In this process, the hydrolysis of the GTP is considered to provide a force in cell division, and the transition from a straight to a curved conformation is deemed to transfer the mechanical force to the membrane, contributing to the accomplishment of the cell division [54,55]. The binding geometry of GTP can give a rational answer to the fact that the binding of GTP can facilitate the FtsZ polymerization and the GTP hydrolysis is related to the depolymerization of FtsZ [40]. In the following years, some researchers have found that the FtsZ-mutant cells without detectable GTPase activity also have the ability of cell division, making the physiological role of division force controversial, and the GTP hydrolysis is not regard as the source of power during constriction of the septum, but a promotive factor in symmetric invagination of the cell division [56,57]. In fact, it is membrane fluctuation which finishes the membrane division in the final step and the remodeling of the cell wall offers the positive force, accelerating the septum toward closure, and then leading to the final division [58e60]. The assembly of FtsZ subunits occurs in a temporally-controlled stage-specific manner at the mid-cell. Several regulatory factors, such as Min-CDE system and Noc (Bacillus subtilis)/SIma (E. coli), can guarantee the location of assembling between the segregated chromosomes in the mid-cell rather than at the cell poles [58,61]. The lateral interactions among protofilaments are regarded as the result of the protofilaments annealing by some researchers [58]. However, others have proposed that the interactions may be due to the electrostatic forces involving the ions between the protofilaments [52,62]. To date, the mechanism of them remains to be in dispute. In the formation of Z-ring, FtsZ-interacting proteins play an essential role. Among them, the most important proteins are FtsA and ZipA, through which FtsZ is attached to the cell membrane. The two proteins bind to the conserved C-terminal domain of FtsZ, necessary for proper cytokinesis [23,63]. As FtsA can replace ZipA

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by a mutation and has a widely conserved scope compared with ZipA, it is considered to be a more significant factor responsible for Z-ring integrity than ZipA [24,61]. Additionally, the Z-ring formation is affected by the other stabilizing proteins such as ZapA and SepF, and the destabilizing proteins such as SulA, EzrA and MinCD [32,64], as well as some other factors like glutamate [65], calcium [66,67], pH value and the ionic strength [68]. Thus, the process in which FtsZ assembles into Z-ring is precisely tuned. Besides the control of accessory proteins for the spatial organization of Z-ring, the intracellular FtsZ polymerization also can be used for the control of Z-ring formation [18]. Bacteria regulates Zring organization through the accurately administration of the intracellular GTP-FtsZ concentrations which are mainly controlled by the rate of nucleotide turnover [69]. It is reported that the nucleotide turnover rate can vary from 6.9 nmol mg
1 h
1 for Mycobacterium tuberculosis (M. tuberculosis) to 30 mmol mg
1 h
1 for E. coli [69]. However, the GTPase activity of FtsZ has not been found to be essential in cell division [56] and the GTP functions in Zring dynamics remain to be unclear although the GTP-binding has a promotive effect on the stability of protofilament [49]. Moreover, the specific amino acid residues responsible for the nucleotide hydrolysis are not located in the GTP-binding region, but in the T7 loop on the opposite side of the FtsZ. It further proves the head-totail association manner of individual FtsZ monomers, which keeps in line with the reported FtsZ crystal structure of Methanococcus jannaschii (PDB: 1W5A) [57]. 3. FTSZ inhibitors 3.1. Natural products and semi-synthetic compounds Cinnamaldehyde (1), 3-phenyl-2-propenal, is a natural aromatic compound derived from the stem bark of Cinnamomum cassia. It has been reported to exhibit broad-spectrum antibacterial activity with MIC values of 1 mg mL
1 for E. coli, 0.5 mg mL
1 for B. subtilis and 0.25 mg mL
1 for MRSA [70]. However, Domadia et al. [70] recently determined the antibacterial activity of cinnamaldehyde against E. coli and B. subtilis with the MIC values of 1000 and 500 mg mL
1, respectively [70]. The obvious difference could be due to the volatile loss and instability of this compound in vivo [70]. The administration of this compound showed that the light scattering intensity of FtsZ protein increased in a light scattering assay and the morphology of FtsZ protofilaments became thinner and shorter in electron microscopy images. Cinnamaldehyde against E. coli FtsZ polymerization and GTPase activity was also detected with the IC50 values of 6.86 ± 2.2 and 5.81 ± 2.2 mM, respectively. The STD-NMR spectroscopy [43,47,48,54,71] and in silico docking model with AutoDock software indicated that the binding pocket of cinnamaldehyde was around the T7 loop in the C-terminal region of FtsZ [70]. Viriditoxin (2), originated from Aspergillus viridinutans, was first reported in 1971, but its structure was incorrectly assigned at that time and was corrected in 1990 [72,73]. This compound inhibited E. coli FtsZ polymerization with an IC50 value of 8.2 mg mL
1 and GTP hydrolysis with an IC50 value of 7.0 mg mL
1 [74]. In the morphological assays, viriditoxin could cause the filamentation of cells [74]. Additionally, the induction of FtsZ expression could lead to the increased MIC value, proving the interaction of viriditoxin with FtsZ [74]. Moreover, viriditoxin displayed potent antibacterial activity against many clinically relevant Gram-positive pathogens, such as various strains of S. aureus (MIC ¼ 4
8 mg mL
1), Enterococcus faecalis and Enterococcus faecium (MIC ¼ 2
16 mg mL
1) [74]. The broad-spectrum character of viriditoxin may be resulting from the highly conserved structure of FtsZ in the viriditoxin-binding site [75]. Viriditoxin could be used both as a lead compound for

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the development of broad-spectrum antibacterial agents in the future and as a tool for the further biological studies of FtsZ. Totarol (3), isolated from Podocarpus totara, is a diterpenoid phenol antibacterial agent against a variety of Gram-positive bacteria [76]. Its mechanism of action remained ambiguous until 2007 when Jaiswal et al. [77] revealed that totarol inhibited bacterial cytokinesis by perturbing the assembly of FtsZ using sedimentation assay, GTP hydrolysis assay and electron microscopic analysis. However, in the TNPGTP-replacing assay, this compound had no influence on the binding of GTP to M. tuberculosis FtsZ [77]. Thus, they inferred that the inhibition of assembly possibly resulted from the conformational changes in FtsZ upon ligand binding, hindering the new monomers to the polymer, or failure in the binding of FtsZ to an accessory protein [77]. Besides, totarol displayed a potent activity against B. subtilis 168 and M. tuberculosis with MIC values of 0.6 and 16 mg mL
1, respectively. Interestingly, it could reduce the MIC value of methicillin from 32 mg mL
1 to 4 mg mL
1 [78]. In the binding assay, it was found to share a modest affinity at M. tuberculosis FtsZ (Kd ¼ 11 ± 2.3 mM) [77]. However, it did not affect the growth of Gram-negative bacteria and fungi. Dichamanetin (4) and 2000 -hydroxy-500 -benzylisouvarinol-B (5) are two polyphenolic compounds found independently by Hufford and Anam from Uvaria chamae and Xylopia Africana, respectively [79,80]. The two natural products exhibit antibacterial activity against a variety of pathogens. Especially, they display strong antibacterial activity against Gram-positive bacteria, such as S. aureus (MIC ¼ 0.80 mg mL
1, 1.16 mg mL
1, respectively) and B. subtilis (MIC ¼ 5.01 mg mL
1, 1.77 mg mL
1, respectively), similar to clinically relevant antibiotics [79,80]. As the mechanism of the two compounds was unclear, Urgaonkar et al. [81,82] evaluated their effects on E. coli FtsZ GTPase activity and found their potent inhibition with the IC50 values of 12.5 mM and 8.3 mM, respectively. Chrysophaentins A-H, eight new antimicrobial natural products, were extracted from the marine chrysophyte alga Chrysophaeum taylori by Plaza et al. [83] in the recent years. Among them, chrysophaentin A (6) was the most potent member that blocked the growth of many clinically relevant bacteria, such as MRSA, multidrug-resistant S. aureus, and VRE with MIC50 values of 1.5 mg mL
1, 1.3 mg mL
1 and 2.9 mg mL
1, respectively. Interestingly, the representative chrysophaentin A was found to share the ability to inhibit the GTPase activity of recombinant E. coli FtsZ with an IC50 value of 6.7 mg mL
1 and block the GTP-induced formation of FtsZ protofilaments. Further STD-NMR experiments and NMR competition experiments with GTPgS confirmed the competitively binding of chrysophaentin A to the GTP binding site of FtsZ. As the continuation of this investigation, Keffer et al. [84] identified other two new linear chrysophaentin analogs, E2 (8) and E3 (9), and accomplished the synthesis of chrysophaentin fragments 10 and 11. Both fragments displayed comparable activity against S. aureus UAMS-1 (a clinical osteomyelitis isolate) and CA-MRSA USA300LAC with MIC values ranging from 1 to 10 mg mL
1, similar to the linear chrysophaentins E (7), E2 and E3. Furthermore, Keffer et al. [85] also conducted the GTPase assays for the target confirmation of 10. In the GTPase assay, 10 exerted the inhibition against the hydrolysis activity of E. coli FtsZ with an IC50 value of 37 ± 7 mM and S. aureus FtsZ with an IC50 value of 38 ± 9 mM. The fluorescence anisotropy was used for the study of the interaction between 10 and FtsZ to indicate a competitive binding of 10 with GTP to the nucleotide binding region of FtsZ. Berberine (12), as a natural plant alkaloid, is obtained from various species of Berberis, such as Berberis aquifolium and Berberis aristata [86]. It exhibits antibacterial activity against a number of pathogenic Gram-positive and -negative bacteria [87,88]. Furthermore, berberine is active against multiple drug-resistant M. tuberculosis [89] and MRSA [90]. Domadia et al. [86] adopted a

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series of biochemical assays, NMR and fluorescence spectroscopy, modeling electron and confocal imaging and ITC to investigate its antibacterial mechanism. Consequently, berberine inhibited FtsZ assembly with an IC50 value of 10.0 mM in the light scattering assay and disturbed GTPase activity in a dose-dependent manner with an IC50 value of 16.01 mM. The result of its group epitope mapping with FtsZ demonstrated that its dimethoxy groups, isoquinoline nucleus and benzodioxolo fragment interacted closely with FtsZ. Together, in silico docking study with AutoDock software indicated that the proposed putative binding site was in agreement with the STD-NMR data. The imaging analysis displyed that berberine could affect Z-ring morphology by disturbing its typical midcell localization and reduce the frequency of Z-rings per unit cell length with a percentage of 50%. Additionally, the ITC showed that the interaction of berberine with FtsZ was driven by entropy with a dissociation constant of 0.02. Berberine is verified to be an FtsZ-targeted naturally occurring compound with elucidated mechanism, which will lay the foundation for the exploration of specific antibacterials targeting FtsZ through rational drug design. Curcumin (13) is a dietary polyphenolic compound, found in the rhizomes of Curcuma longa. It exhibits antibacterial activity against a variety of pathogens, including Gram-positive and -negative bacteria [91]. In morphology, it can induce cell filamentation in B. subtilis 168, which suggests that this compound may inhibit bacterial cytokinesis. Dipti R. et al. [92] thought that the target of curcumin could be FtsZ and proved their hypothesis with the microscopy analysis, light scattering assay and GTPase activity assay. The electron microscopic analysis indicated that curcumin inhibited Z-ring formation by perturbing FtsZ assembly and bundling. In light scattering assay, curcumin decreased the light scattering intensity of FtsZ protofilaments, indicating its inhibiting the polymerization of FtsZ. In the GTPase assay, curcumin increased the GTP hydrolysis rate. For example, 30 mM of curcumin could increase the GTPase activity of FtsZ by 35% compared with the control [92]. In the extensive study using computational docking [93], curcumin was speculated to bind a pocket in which most residues were found to be involved in GTP-binding as well. Curcumin, however, had poor bioavailability and influenced the mammalia microtubule Fig. 1. 3.2. Synthetic small molecules 3.2.1. From available chemicals Since rhodanine compounds like OTBA were reported to have antibacterial activity by inhibiting bacterial growth via influencing FtsZ [94], Singh et al. [95] screened an array of rhodanine analogues to find novel FtsZ-targeted antibacterial agents. Among the tested compounds, CCR-11 (14) could exert inhibition against the growth of B. subtilis cells by more than 50% at a concentration of 2 mM and could induce the elongation of B. subtilis cells in the morphology. Subsequently, results from some molecular assays such as GTPase assay and light scattering assay showed that CCR-11 could arrest the FtsZ GTPase activity and FtsZ polymerization. The effects on Zring and nucleoids as well as membrane were further checked in elongated B. subtilis. The results indicated that the nucleoids as well as membrane were visibly free from CCR-11 while Z-ring formation was seriously perturbed with a reduced frequency of occurrence. The docking study predicted that CCR-11 bound in a cavity of FtsZ adjacent to the T7 loop. And the theoretically estimated binding energies were similar to the experimentally determined binding constant (1.5 mM), further confirming the docking results. With respect to the selectivity of CCR-11, the influences of this molecule on HeLa Cells showed that it exhibited weak activity against the mammalian cells with an IC50 value of 18.1 ± 0.2 mM. Since the previous reported that GTP analogs could inhibit FtsZ polymerization without affecting tubulin assembly [96], Ruiz-Avila

et al. [97] developed a mant-GTP fluorescence anisotropy competitive assay to screen a collection of compounds from the literature, virtual screening hits and in-house compounds. Interestingly, inhouse compounds screened gave several good compounds such as UCM05 (15), its fragments UCM16 (16) and UCM17 (17) with significant affinities. Additionally, their analogues, such as UCM44 (18) and UCM53 (19), were identified with stronger affinities. Treatment of B. subtilis FtsZ with UCM05 and UCM44 resulted in the reduced light scattering as well as the amount of sedimentable polymer. However, a weak (20%) inhibition of both compounds against the GTPase activity was observed in the GTPase assay. In the electron microscopy analysis, the reduced lateral association of E. coli FtsZ filaments with the treatment of UCM05 and UCM44 was obvious with respect to the controls. In the cellular experiment, UCM05, UCM44 and UCM53 displayed activity against various bacteria (Table 1). The fused FtsZ-GFP was employed to visualize the Z ring formation in B. subtilis SU570 cells. The result showed that these compounds induced formation of increased punctuate foci and un-normal ring numbers, indicating a functional impairment of FtsZ. Additionally, the effects of these compounds on mammalian cells demonstrated a partially overlapping inhibition concentrations with the bacterial MICs, and among them, UCM53 displayed a best selectivity [IC50 (HeLa)/MIC (S. aureus MR 12160636) ¼ 5]. Due to the great potential of natural sources as well as the popularity of in silico screening [98e100], Chan et al. [101] carried out a structure-based virtual screening of large natural compound libraries from Analyticon Discovery. In this study, ten compounds showed the GTP-like contacts with the enzyme (M. jannaschii FtsZ, PDB entry 1W5B) and were selected for the further biochemical evaluations. The results suggested that 20 possessed activity in both GTPase activity assay and antimicrobial susceptibility assay (Table 2), while the other nine compounds were inactive. In the induced-fit docking, 20 simulated the native interactions of GTP substrate with GTP binding region. Homology modeling of S. aureus FtsZ was also used for screening new quinuclidine derivatives, leading to the discovery of a new series of compounds. Among them, compounds 21, 22 and 23 exhibited significantly improved activity in the GTPase activity assay and antimicrobial susceptibility assay. Especially, 23 displayed more potent activity than totarol in the GTPase activity assay and exerted dramatically enhanced antibacterial activity against the Gram-negative strain of E. coli compared with totarol. Moreover, compound 23 exhibited no effects on the tubulin polymerization, suggesting its good selectivity against FtsZ. Since DAPI (24), as a fluorescence probe, was substantiated to bind to the main body (tubulin S) of tubulin with a high affinity [102e104], it was further investigated for the effects on E. coli FtsZ [105]. In the light scattering assay, DAPI induced the enhancement of light scattering in a concentration-dependent fashion in the absence of GTP, and after the addition of GTP, the enhancement of light scattering over the initial scattering was observed. The GTPase assay further showed the decreased maximal velocity of the GTPase activity and the reductive MichaeliseMenten constant of 29.4 ± 0.3. The fluorescence anisotropy was measured in the titration of DAPI, giving the dissociation constant with a value of 16.6 mM. In the electron microscopic morphology analysis, DAPI exerted the positive effects on the width of FtsZ filaments in a concentrationdependent manner, indicating the stabilization on the protofilament bundles. Although it is admitted that most aminoglycosides display their function via inducing codon misread in the process of protein synthesis, the precise mechanisms on the inhibition of pathogens remain to be poorly understood [106,107]. In order to unravel the cell processes especially susceptible to aminoglycosides and to

X. Li, S. Ma / European Journal of Medicinal Chemistry 95 (2015) 1e15

5

Fig. 1. The structures of the natural products.

understand the mechanisms of their action preferably, Possoz et al. [108] undertook a project at a cellular level to investigate the cell elongation and division as well as the dynamics of chromosome replication and segregation in E. coli cells with the treatment of Table 1 Inhibition of bacterial cell growth MIC (mg mL
1) [97]. Microorganism B. subtilis 168 S. aureus methicillin-S ATCC 29213 E. faecium ampicillin-R and levofloxacin-R 12160560 E. faecalis ATCC 29212 S. pneumoniae ATCC 49619 A. baumannii multi-R 12159796 P. aeruginosa ATCC 27853 E. cloacae multi-R 12161389 E. coli ATCC 35218 K. pneumoniae e ESBL ATCC 700603

UCM05 (15)

UCM44 (18)

sublethal concentrations of amikacin (25). This drug at concentration of 4 or 8 mg mL
1 resulted in the cells with longer sizes than the normal. Subsequently, the ectopic FtsZ-cyan fluorescent protein fusion technique was applied to the E. coli cells for observing the behavior of FtsZ. With the treatment of this antibiotic, the percentage of cells containing Z ring was very low (35%) in sharp contrast to that of untreated cells (88%) and the FtsZ signals were

UCM53 (19)

64 32 64

16 16 32

4 8 4

64 64 64 128 128 128 128

32 32 64 64 128 128 128

2 32 32 64 128 128 128

Table 2 FtsZ GTPase inhibition and antimicrobial activity of pyrimidine substituted quinuclidines and totarol [101]. Compounds

IC50(mM)

20 21 22 23 Totarol

317.2 73.2 55.7 37.5 40.8

± ± ± ± ±

17.0 4.1 2.4 2.2 15.1

MIC(mg$mL
1) S. aureus

E. coli

384.5 24.0 12.0 9.0 1.5

192.4 36.0 72.0 18.0 >400.2

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distributed throughout the cytoplasm, exerting the intervention of the Z ring formation. Amikacin was preliminarily confirmed to have the ability to influence the function of FtsZ, indicating the appearance of another tool drug for the investigation of FtsZ characteristics Fig. 2. 3.2.2. From rational drug design 3.2.2.1. 3-alkoxylbenzamides. 3-Methoxybenzamide (3-MBA) (26) was reported to serve as an inhibitor of cell division via targeting FtsZ in the Gram-positive bacterium B. subtilis, leading to a filamentous morphology [109]. Considering the advantages of 3-MBA such as small molecular mass, good ligand efficiency with on-target activity and nice cell penetration, Haydon et al. [6] carried out a medicinal chemistry program to design and synthesize various 3MBA derivatives. In particular, PC190723 (27) was found to possess potent activity against all the tested staphylococci with an MIC value of 0.5e1.0 mg mL
1. However, it showed no activity against a range of Gram-negative pathogenic bacteria as well as the yeast or human hepatocytes. Furthermore, PC190723 displayed

significant inhibition against GTPase activity of S. aureus FtsZ with an IC50 value of 55 ng mL
1 leading to the mislocalization of FtsZ in the constructed B. subtilis containing GFP-FtsZ. In the cell morphology, PC190723-treated rod-shaped B. subtilis or spherical S. aureus exerted the elongation or enlargement of the cells, respectively. The cooperative results substantiated that the direct interaction of PC190723 with FtsZ resulted in the interruption of septum formation and then the cell division. The docking study indicated that the ligand bound to the cleft between helix seven and the C-terminal domain, keeping in line with the location of mutant residues. Therefore, PC190723 represents a novel class of FtsZ inhibitors with potent activity, and is amenable to further optimization for the treatment of staphylococcal infection. Stokes et al. [110] carried out a further optimization of 3methoxybenzamide derivatives in order to achieve better candidate drugs. They replaced the bicyclic thiazolopyridine moiety of PC190723 with a substituted phenyl bromo-oxazole moiety to give a promising compound 28 that possessed an average MIC value of 0.12 mg mL
1 against all the staphylococcal species, showing 4- to

Fig. 2. The structures of the available chemicals targeting FtsZ.

X. Li, S. Ma / European Journal of Medicinal Chemistry 95 (2015) 1e15

32-fold more potent activity than PC190723. Similar to PC190723, 28 was observed to have little or no activity against the other pathogens tested. The phenotype of S. aureus ATCC 29213 was analyzed, appearing enlarged and multinucleate cells with the treatment of 28 at a wide concentration range beginning at 0.12 mg mL
1. Additionally, compound 28 possessed potent activity against PC190723-resistant S. aureus G196A mutant strain. Compared with 28, its S-enantiomer showed about 100-fold weak activity in the antibacterial assay [111]. The tremendous difference was interesting, which led Chiodini et al. [112] to investigate the configuration and activity of their representative benzodioxanebenzamide derivative 29. Their study revealed that the S-enantiomer displayed 4
5-fold more potent than the R-congener, suggesting the enantiomer switch as an interesting chance for further optimization in the development of FtsZ inhibitors. As the solubility limitation for the in vivo evaluation at higher concentrations, compound 30, a succinate prodrug of 28, which was designed and synthesized, presented the similar stability as well as bioavailability of PO administration at the same drug concentration to that of 28. Compound 30 was subjected to the standard inoculum murine model of staphylococcal thigh infection, showing an obvious inhibition in bacterial burden compared with the control. The above two compounds displayed excellent in vitro and in vivo efficacy, and drug-like properties, presenting their promising potential to be developed into first-in-class agents targeting FtsZ for the treatment of various drug-resistant staphylococcal infections. Stokes et al. [111] continued to investigate the oxazolebenzamide derivatives, designing and synthesizing various analogues. The MIC assays with S. aureus ATCC 29213 revealed that these novel oxazole-benzamide derivatives such as 31 displayed the dramatically enhanced on-target activity against the wild-type S. aureus with an MIC value of as low as 0.03 mg mL
1. Moreover, some derivatives such as 32 possessed antibacterial activity against the previous benzamide-resistant S. aureus G196A mutant. However, the representatives exhibited unsuitability in vivo pharmacokinetic properties, such as clearance rates of 137 mL min
1$kg
1 and the area under the curve (AUC)  0.4 mg h$mL
1. In view of the above-mention situation, several pseudo-benzylic substituted derivatives were prepared and evaluated their pharmacokinetic profiles. Fortunately, every pseudobenzylic substituted derivative displayed ideal AUCs, half-lives and clearance rates, especially, the hydroxymethyl-substituted racemics 33. Furthermore, they had an improved metabolic stability. In view of the potent antistaphylococcal activity and poor druglike properties of the benzamide derivative PC190723, Kaul et al. [113] explored the more druggable benzamide analogs and identified a prodrug of PC190723, designated as TXY541 (34). In the solubility assay, TXY541 was 143-times more soluble than PC190723 in acidic citrate vehicle, which was sufficient in magnitude for its next in vivo efficacy evaluation. The chemical stability of TXY541 after 24 h of incubation in the same vehicle was assessed to be stable using reverse-phase HPLC. PC190723 was detected in staphylococcal growth media and in 100% filtered mouse serum due to hydrolysis of TXY541. The experiment gave a half-life of 8.2 ± 0.4 h in the staphylococcal growth media while 3.4 ± 0.2 min in the 100% filtered mouse serum. The conversion depended on not only pH conditions, but also some enzyme in serum. The antistaphylococcal evaluation indicated that TXY541 displayed identical activity to PC190723 in the presence of mouse serum. However, this compound possessed less potency than PC190723 in the absence of mouse serum, which owed to its slow conversion without the serum. Additionally, the MTT experiment showed an IC50 value of >128 mg mL
1, 64-fold higher than its MIC value, showing a significant therapeutic window. Additionally, a patent application by Lavoie et al. [114] has

7

disclosed a series of PC190723 derivatives that are highly soluble and may be formulated for administration as antibiotic drugs in the future. These derivatives shared the scaffold of PC190723, most of which were characterized by the amino hydrogen(s) substituted with different acyl derivatives in structure. The further pharmaceutical investigation of these compounds may provide promising FtsZ inhibitors with ideal properties Fig. 3. 3.2.2.2. Benzopyridines. Parhi et al. [115] set out to study the effects of aryl substituents at the 2- and the 12-position of berberine derivatives on the antibacterial activity. The resulting data suggested the presence of biphenyl substituent at either 2- or 12-position of berberine derivatives dramatically contributed to its antibacterial potency against S. aureus and E. faecalis. In this test, 2-biphenyl substituted compound 35 exerted the best activity against the methicillin-sensitive and -resistant strains of S. aureus and vancomycin-sensitive and -resistant strains of E. faecalis (Table 3). Subsequently, 35 and 36 were subjected to FtsZ polymerization assessment so that they significantly increased the light signal compared with the control in the light scattering assay, indicating their stimulation of FtsZ polymerization. Additionally, the increased light scattering degree of these two compounds was corresponding to their antibacterial activity. On the basis of the fact that the introduction of a hydrophobic substituent to the structure of sanguinarine or berberine dramatically enhanced antibacterial activity [115], Kelley et al. [116] further simplified their common scaffold to form a new 3phenylisoquinoline core. Subsequently, 3-phenylisoquinolines and 3-phenylisoquinolinium derivatives were synthesized and evaluated for their antibacterial activity. As a result, the quaternary isoquinolinium derivatives exhibited more potent activity than their corresponding non-quaternary isoquinolines and the lipophilicity of the substituent at the 30 -position apparently contributed to the antibacterial efficacy. In the evaluation, compounds 37, 38, 39 and 40 exerted the best potency with MIC values ranging from 1 to 8 mg mL
1 against all the tested strains (Table 3). Moreover, the binding of the isoquinolines to S. aureus FtsZ was monitored using the intrinsic fluorescence of the compounds, giving dissociation constants ranging from 1 to 10 mmol. Furthermore, the representative compounds showed minimal cross-reaction mammalian b-tubulin as well as little or no cytotoxicity to mammalian cells. From the view of medicinal chemistry, this work successfully accomplished the structural simplification of natural products and accelerated the discovery of novel nontoxic anti-FtsZ antimicrobials Fig. 4. 3.2.2.3. Benzopyrazine, benzopyrimidine and pyridinopyridines. In view of the previous reports that some phenyl substituted naphthalenes and isoquinolines possessed the potential antibacterial activity via targeting FtsZ [116e118], Parhi et al. [119] designed and synthesized a series of phenyl substituted quinoxalines, quinazolines and 1,5-naphthyridines, and evaluated their activities with the aim to discover the novel anti-FtsZ agents. The resulting data showed that some compounds displayed comparable activity, such as quinoxaline derivatives 41, 42, 43, 44 and 45, quinazoline derivative 46 and 1,5-naphthyridine derivative 47 (Table 4). Furthermore, in the light-scattering assay, the nonquaternary compounds 44, 45, 46 and 47 exerted the stimulation of FtsZ polymerization, and in sharp contrast to them, the quaternary quinoxaline derivatives 41 and 42 were found to have no significant influence on FtsZ polymerization. With the goal of discovering the useful and reliable inhibitors of FtsZ, Anderson et al. [120] conducted a broad biochemical crosscomparison of known FtsZ inhibitors and then selected some reliable molecules for their further search for the better inhibitors. In

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Fig. 3. The structures of 3-alkoxylbenzamides.

their comparison, zantrin Z3 was selected and its preliminary modifications led to the discovery of compound 48, which possessed the best activity in this series with an IC50 value of 12 mM for the inhibition of E. coli FtsZ. Further investigation of this compound, such as cocrystallization with B. subtilis FtsZ and E. coli FtsZ as well as their isolation of resistant strains of E. coli and B. subtilis was under way Fig. 5. 3.2.2.4. Pyridopyrazines and pyrimidopyrazines. Considering the FtsZ protein serving as the distant relative of eukaryotic tubulin, Reynolds et al. [121] not only screened their in-house antitubulin compounds, but also the antimalarial drugs with the similar structures to the antitubulin molecules [122] in order to find the anti-FtsZ agents. Their investigation led to the discovery of 3deazapteridine compounds 49 and 50 with active effects on M. tuberculosis FtsZ as well as M. tuberculosis strain H37Ra (Table 5). As a continuation, Mathew et al. [123] synthesized pyridopyrazine and pyrimidothiazine analogs based on the structures of the above two compounds to improve their potency against FtsZ, to increase antibacterial activity, and to reduce off-target toxicity. In their project, all the newly prepared compounds were evaluated against

M. tuberculosis H37Ra, M. tuberculosis H37Rv and Vero cells. The cellular results showed that most of compounds possessed good antibacterial activity, while concurrently shared cytotoxicity to Vero cells. In the molecular test, a majority of compounds exerted inhibition of FtsZ without any effects on tubulin. Taking all the results into consideration, the synthetic compound 51, sharing the similar properties to 49, presented the ideal characteristics (Table 6). In the short term Interferon-cgene-disrupted C57BL/6 mice (GKO) model, 49 displayed significant efficacy at 10 mg kg
1 in the lungs, reducing the bacterial load by 0.86 Log 10 CFU (P < 0.05), whereas no appreciable impacts in the spleen. As for 51, no detectable efficacy was found in lungs as well as spleens after 9 days of treatment at the same dosage. Due to the broad toxicity as well as the other critical issues in this series, Mathew et al. eventually deemphasized this class of compounds and started to pursue other FtsZ inhibitors from the screening Fig. 6. 3.2.2.5. Benzimidazoles. Enlightened by the structural similarity of the previously reported pyridopyrazines, pteridines, albendazole and thiabendazole, which shared effective anti-FtsZ activity, Kumar et al. [124] designed and synthesized a library of novel molecules

Table 3 Antistaphylococcal and antienterococcal activities of 3-phenyl-6,7-dimethoxyisoquinoline and 3-phenyl-6,7-dimethoxy-2-methylisoquinolinium derivatives synthesized [115,116]. Compounds

MIC(mg mL
1) S. aureus 8325-4 (MSSA)

S. aureus ATCC 33591 (MRSA)

E. faecalis ATCC 19433 (VSE)

E. faecalis ATCC 51575 (VRE)

35 37 38 39 40

0.5 1 1 2 2

0.5 2 1 2 2

2 4 4 8 8

2 8 4 8 8

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9

Fig. 4. The structures of benzopyridines.

with benzimidazole scaffold for the development of novel FtsZ inhibitors. All the newly synthetic compounds were evaluated for their activity against M. tuberculosis H37Rv using the microplate Alamar Blue assay (MABA) [125]. Of these compounds, 11 compounds were confirmed to show MIC values of 0.39e6.1 mg mL
1 without any appreciable toxicity (IC50 > 200 mM) against Vero cells. Among them, compounds 52, 53, 54, 55 and 56 were selected as the representatives for the further assessment against the clinical isolates of M. tuberculosis strains with different drug-resistance profiles. Fortunately, the five compounds displayed the similar efficacy against the drug-resistant strains to that against the M. tuberculosis H37Rv (Table 7). At molecular level, they presented the inhibition of FtsZ polymerization to different degrees, and exhibited the unexpected enhancement of GTPase activity, similar to the effects of curcumin. Additionally, TEM and SEM analyses implied that they also effectively perturbed the density, length and thickness of FtsZ protofilaments, as well as the cell division lengths and septum formation. Subsequently, Knudson et al. [126] selected 53, 54 and 57 from this series for the further activity studies, resulting in the in vivo efficacy and in vitro-in vivo activity relationship of 2,5,6trisubstituted benzimidazoles demonstrated firstly. This work is of importance in confirming the prospect of trisubstituted benzimidazoles for the discovery of antimycobacterial agents targeting

FtsZ. In the recent years, Awasthi et al. [127] carried out the further systematic modification of the previously reported lead compounds 53 and 54 by optimizing the nitrogen substituents at the 5 and 6 positions and keeping the cyclohexyl group at the 2 position intact concurrently. This study led to the discovery of more than 10 newly identified compounds with an MIC value below 1 mg mL
1 against M. tuberculosis H37Rv strain, such as compounds 58, 59 and 60. Compounds 64 and 60 were determined for the activity against clinical isolates of M. tuberculosis possessing different resistance profiles, the MIC values of which were identical to those for sensitive M. tuberculosis H37Rv strain. Moreover, the light scattering assay indicated that compounds 58, 61 and 60 reduced the light scattering obviously, suggesting their inhibitory effects on M. tuberculosis FtsZ assembly. As the supplementary proof, TEM imaging demonstrated that 58 not only significantly reduced the density and population of FtsZ polymers, protofilaments and aggregates, but also apparently disrupted the formed FtsZ polymers and aggregates. All the results together suggested that the above trisubstituted benzimidazoles might exert their activity through their interaction with M. tuberculosis FtsZ. To further investigate its durg-like properties, Knudson et al. [128] set out a program to study the stability and in vivo susceptibility of the representative

Table 4 Antibacterial activities of quinoxaline, quinazoline and 1,5-naphthyridine derivatives [119]. Compounds

41 42 43 44 45 46 47

MIC(mg$mL
1) S. aureus 8325-4 (MSSA)

S. aureus ATCC 33591 (MRSA)

E. faecalis ATCC 19433 (VSE)

E. faecalis ATCC 51575 (VRE)

0.5 2.0 0.5 4.0 4.0 4.0 8.0

0.5 2.0 1.0 4.0 4.0 8.0 8.0

8.0 8.0 16 8.0 8.0 32 32

8.0 16 16 16 16 32 64

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Fig. 5. The structures of Benzopyrazines, benzopyrimidines and pyridinopyridines.

Table 5 Activities of compounds 49 and 50 [121]. Compounds

49 50

MICs (mg$mL
1, for M. tuberculosis H37Ra)

0.25 [164] 2

M. tuberculosis FtsZ

Tubulin

Polymerization ID50 (mM)

GTP hydrolysis

Polymerization (100 mM)

34.2 ± 2.5 38.1 ± 4.1

35% at 10 mM 23% at 20 mM

No inhibition 24%

Table 6 Activities of compounds 49 and 51 [123]. Compounds

M. tuberculosis FtsZ polymerization IC50(mM)

Tubulin polymerization IC50(mM)

M. tuberculosis H37Ra MIC(mg$mL
1)

M. tuberculosis H37Rv IC90(mM)

Vero cytotoxicity CC50(mM)

49 51

34.2 ± 2.5 34.3 ± 6.0

>100 mM >100 mM

0.25 0.23

400 >400 >200 >200 >200

Fig. 7. The structures of benzimidazoles.

Table 8 Antistaphylococcal and antienterococcal activities of 4- and 5-substituted 1phenylnaphthalenes [117,118]. Compounds

64 65 68 69 70

MIC (mg$mL
1) S. aureus 8325-4 (MSSA)

S. aureus ATCC 33591 (MRSA)

E. faecalis ATCC 19433 (VSE)

E. faecalis ATCC 51575 (VRE)

0.5 0.5 2.0 0.5 1.0

0.5 0.5 2.0 2.0 2.0

2 2 2.0 2.0 2.0

2 2 2.0 4.0 2.0

polymerization, while 64 appeared little or no effects on the light scattering of porcine b-tubulin. The simplified analogues of sanguinarine and chelerythrine,

compounds 67 and 63 that were benzo[c]phenanthridines bearing hydrophobic functionality at either 12- or 1-position, were found to exert the enhanced antibacterial activity [130]. However, the constitutive cationic charge on their structures contributed to an adverse affection on their desired pharmacokinetic properties. Based on the facts, Kelley et al. [117] designed and synthesized an array of 4- and 5-substituted 1-phenylnaphthalenes, which not only shared a truncated form within their core structures, but also lacked the constitutive cationic charge. All of the compounds showed significant antibacterial activity against the methicillinsensitive and -resistant S. aureus as well as vancomycin-sensitive and -resistant E. faecalis with MIC values of 0.5e16 mg mL
1. Comprehensively, compounds 68, 69 and 70 displayed the optimal antibacterial activity against all the tested strains (Table 8). Subsequently, in the FtsZ polymerization assay, as expected, compounds 71 and 69 significantly increased the kinetics and extent of S. aureus FtsZ polymerization. These preliminarily results indicated

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that the antibacterial activity of these compounds might attributed to their disturbance of FtsZ polymerization. This program improved the physico-chemical properties and the activity of the previous alkaloids and their analogues, favoring their in vivo absorption and distribution Fig. 8.

3.2.2.7. Others. As the continuation of the previously discovered heterocyclic molecules, such as benzo[c]phenanthridines and dibenzo[a,g]quinoliziniums [115e130], Kaul et al. [10] identified another series of heterocyclic compounds bearing a guanidinomethyl biaryl scaffold, which could increase the FtsZ selfpolymerization and possess potent bactericidal activity. In the binding affinity assay, compound 72 displayed Kd values of 3.5 ± 0.5 mM and 3.8 ± 0.3 mM, respectively, in its interactions with S. aureus FtsZ and E. coli FtsZ. While its Kd value in the corresponding interaction with E. faecalis FtsZ significantly increased to 21.8 ± 4.6 mM. In the examination of self-polymerization activity, 72 acted as a stimulator for the polymerization dynamics of all the three FtsZ proteins of the S. aureus, E. coli, and E. faecalis in a concentration dependent manner. The electron microscopy analyses showed that 72 induced the long filamentous polymers in both proteins, while it triggered polymer widths of 4e25 nm in the S. aureus FtsZ and 4e6 nm in the E. coli FtsZ. The anisotropy of a fluorescent non-hydrolyzable GTP analog indicated that 72 exerted no effects on the interaction of GTP analog with S. aureus FtsZ, E. coli

FtsZ, and E. faecalis FtsZ. Further, the Autodock Vina docking algorithm revealed that 72 bound to S. aureus FtsZ at a pocket near to the previously reported binding site of PC190723 [6], in which the residues E185 and I228 formed key interactions. Subsequently, the site-directed mutagenesis of the E185 and I228 residues in S. aureus FtsZ verified the computational result. Furthermore, the antibacterial activity of 72 was evaluated against different Gram-positive and -negative bacteria (Table 9) exhibiting the minimal potential to induce resistance in S. aureus. Compound 72 exerted the promising properties as a lead compound to develop novel agents against known MDR bacterial pathogens. Previous study showed that vanillin possessed antimicrobial activity and was used as a natural food preservative [131]. And its Schiff bases also had a broad range of biological activities [132]. Sun and co-workers [133] applied the hybrid drug strategy to design and synthesize two vanillin Schiff base derivatives for their antibacterial evaluation. The resulting data showed that both compounds displayed potent antibacterial activity against E. coli, Pseudomonas aeruginosa fluorescence, B. subtilis and S. aureus. Strikingly, compound 73 possessed an MIC value of 1.56e6.25 mg mL
1 against the tested strains, being much more potent than its congener 74 with an MIC value of 6.25e12.5 mg mL
1. Subsequently, in the FtsZ polymerization assay, 73 displayed strong interruption against the assembly of FtsZ with an ID50 value of 2.1 mM, compared with 74 with an ID50 value of

Fig. 8. The structures of naphthalenes.

X. Li, S. Ma / European Journal of Medicinal Chemistry 95 (2015) 1e15

13

Table 9 Antibacterial Activities of 72 against various Gram-positive and -negative bacteria [10]. Compound

72

MIC(mg$mL
1) S. aureus 8325-4 (MSSA)

S. aureus ATCC 49951 (MSSA)

S. aureus ATCC 33591 (MRSA)

S. aureus Mu3 (MRSA)

E. faecalis ATCC 19433 (VSE)

E. faecalis ATCC 51575 (VRE)

1.0

1.0

1.0

1.0

4.0

4.0

Table 10 Antibacterial activities of synthetic compounds [134]. Compounds

MIC (mg$mL
1) Gram-negative bacteria

75 76 77 78

Gram-positive bacteria

E. coli ATCC 35218

P. aeruginosa ATCC 13525

B. subtilis ATCC 6633

S. aureus ATCC 6538

3.45 21.59 6.12 0.28

5.12 20.83 5.02 5.89

21.58 2.78 2.89 3.31

17.19 6.92 11.34 2.03

Fig. 9. The structures of other compounds targeting FtsZ.

7.6 mM. Moreover, the molecular docking study using Discovery Studio software (version 3.1) indicated that the two compounds docked to the same pocket, different from that of the previously reported molecules. Recently, Sun et al. [135] continued the previous work and accomplished the design and synthesis of an array of vanillin derivatives. Interestingly, a number of the newly synthetic compounds such as 75, 76, 77 and 78 exhibited significant antibacterial activity against both the two Gram-negative bacterial strains (E. coli and P. aeruginosa) and two Gram-positive bacterial strains (B. subtilis and S. aureus) (Table 10). Strikingly, 78 had an MIC value against E. coli, lower than that of the positive control kanamycin. In the FtsZ polymerization assay, 78 exhibited the most potent inhibitory polymerization with an IC50 value of 2.1 mM. In the subsequent docking study with LigandFit Dock protocol of Discovery Studio 3.1, this compound showed the lowest interaction energy and perfectly bound to the FtsZ via five interaction bonds. The potent antibacterial activity as well as the preliminarily confirmed target of the novel vanillin derivatives provided valuable insight into the exploration of FtsZ inhibitors as antibacterial agents Fig. 9.

4. Conclusions Bacterial pathogens, especially the drug-resistant M. tuberculosis, MRSA, VRE and others have greatly threatened human health all over the world [1]. As a powerful respond, a number of antibiotics were developed and marketed in the past three decades [135]. However, most of the drugs are still limited to the classical antibacterials that disturb the biosynthesis of protein, nucleic acid, and cell wall, etc. [136]. Moreover, bacterial resistance has rapidly resulted in many originally powerful antibacterial agents either weak or no activity in clinic, which becomes an increasingly serious concern. To combat the adaptive machinery of bacteria, there is a dire need for the identification of new targets and new pharmacophores for the development of entirely different antibacterial agents, especially with novel mechanisms of action [137]. FtsZ, an essential bacterial cytokinesis protein, exerts its promising potential as an excellent therapeutic target for the discovery of new generation antibacterial agents. Firstly, FtsZ has been confirmed to be indispensable in the bacterial cell division. Any delay or disturbance of FtsZ assembly will result in the failure of the

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bacterial cell division and then the bacterial apoptosis. Secondly, FtsZ is highly conserved in almost all bacteria and is very useful as a target for the discovery of broad-spectrum antibacterial agents, capable of eradicating infections by various bacterial pathogens [103]. Thirdly, though eukaryotic cytoskeleton protein tubulin shares some structural and functional features with FtsZ [138], it is considered to be a distant homologue of FtsZ. For example, some discovered FtsZ inhibitors, such as natural product viriditoxin [74] and synthetic compound PC190723 [6], display potent inhibition against FtsZ without detectable effects on tubulin. Fourthly, The crystal structures of FtsZ from several organisms are available [12,139], which provide not only much information about FtsZ structures, but also an excellent opportunity for structure-guided drug discovery. In total, FtsZ can serve as a promising therapeutic target for the discovery of antibacterial agents with novel mechanism. As reviewed in this paper, many FtsZ inhibitors exhibit perfect potential as the candidates for the further preclinical investigation. Strikingly, the benzamide derivative PC190723 [6] exerts the potent antibacterial activity against various staphylococcal strains and has been confirmed to be efficacious in the animal infection model. As its poor drug-like properties, investigators conducted further pharmacokinetic optimization, leading to some excellent prodrugs [113,114]. For instance, TXY541 [113] exhibits the excellent pharmacokinetic properties. These benzamide derivatives are under further evaluation and are highly possible to go to market as the first FtsZ inhibitor against the staphylococcal infections in the near future. However, most of these compounds exerted poor activity against other Gram-positive and Gram-negative pathogens. For instance, PC190723 inhibited both Streptococcus pneumonia and E. faecalis with MIC values of higher than 64 mg mL
1 [6]. It will be another attempt to discover benzamides targeting FtsZ with broad spectrum. Additionally, the natural products are abundant sources all the time for the drug discovery. In view of sanguinarine [100] and berberine [86] identified as FtsZ inhibitors, the simplification of their structures has produced a series of compounds with MIC value in the levels of submicrograms per milliliter [115e130]. More importantly, the majority of natural compounds possessed broadspectrum antibacterial activity, providing access to developing FtsZ inhibitors with wide antibacterial spectrum. And the development of efficient screening method and target validation technique would accelerate to discover more and more natural compounds targeting FtsZ in this field. Besides, the GTP, as the lead compound, also played a significant role in the discovery of FtsZ inhibitors. For instance, some of its analogues have been found to display a promising potential with good selectivity toward FtsZ [74]. Since FtsZ was a homologue of human tubulin and many antitubulin agents also disturbed the function of FtsZ, the development of anti-tubulin agents provided more chance for the discovery of FtsZ inhibitors. Encouraging results from these explorations warrant that the exploiting FtsZ inhibitors as antibacterial agents are promising and are very likely to emerge in the clinic in the next couple of years. Conflict of interest The authors declare that this study was carried out only with public funding. There is no funding or no agreement with commercial for profit firms. Acknowledgments This research was supported financially by the National Natural Science Foundation of China (20872081 and 21072114), the Natural Science Foundation of Shandong (ZR2010HM092), and

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