A dynamic bacterial cytoskeleton consisting of FtsZ and other proteins is a potential target for the development of antibacterial drugs. GTPase activity of FtsZ protein leads to self-assembly of the protein. The resultant circumferential dynamic Z-ring at the centre of the cell recruits other proteins during progression and completion of bacterial cell division. There are natural compounds inhibiting one or more of these steps. Such inhibition ultimately culminates in the arrest of cell division. In this issue of the Biochemical Journal, a paper by Beuria et al. highlights the importance of the dynamics of the Z-ring for cell division. The ligand-induced enhanced degree of stabilization of FtsZ protofilaments, leading to the absence of the subsequent dissociation step, would hamper the normal functioning of the Z-ring, leading to an inhibition of cell proliferation. A novel antibacterial agent, OTBA (3-{5-[4-oxo-2-thioxo-3-(3-trifluoromethylphenyl)-thiazolidin-5-ylidenemethyl]-furan-2-yl}-benzoic acid) works via this hitherto unreported pathway. It stabilizes the FtsZ polymers, suppressing the dynamics which, in turn, inhibits cell division.

A conventional method of drug discovery has been to search for antibiotics from plant and bacterial sources, screen them for various bacterial infections and employ them if found suitable. More often than not, studies of their specific modes of action have preceeded their use as therapeutics. During the past few decades, semi-synthetic and synthetic compounds based upon the templates of naturally occurring antibacterial agents have also gained prominence in the fight against bacterial invasions. However, in recent times, the failure of commonly used antibiotics to treat infections from bacteria which get the better of us by means of their adaptive genetic machinery is looming large. The emergence of resistant bacteria is a major concern for physicians. In the period spanning the last two to three decades or so, the approach to combat them has taken a new turn. Being enriched with the knowledge of bacterial growth, metabolism and their survival strategy, current efforts have gone to target key metabolic processes for the growth and survival of bacteria for antibacterial drug development. Among them, bacterial cytokinesis, an integral process for the growth of bacteria, has emerged as a major target for the design of novel antibacterial drugs [13]. Over the last two decades a lot of biochemical, biophysical, genetic, ultrastructural and cell-imaging work has been done to throw light on the molecular aspects of the dynamics of bacterial cytokinesis [3]. Different components of the dynamic bacterial cytoskeleton have been identified, along with their functions. Bacterial tubulin (FtsZ), actin (MreB) and intermediate filament (IF) proteins make up a class of proteins which play a vital role in cell division, chromosome and plasmid segregation, and perpetuation of proper cell shape, as well as in maintenance of cell polarity and assembly of intracellular organelle-like structures. Among these proteins, FtsZ protein has been demonstrated to be a key player in cell division [2,3]. Multiprotein assembly consisting of FtsZ protein at the site of cell division causes the constriction of the cytoplasmic membrane, along with various layers of the cell wall [4,5].

FtsZ is a structural homologue of tubulin consisting of a fold akin to that of a tubulin dimer made up from α- and β-tubulin. The crystal structure of FtsZ from the thermophilic bacterium Thermotoga maritima and phylogenetic analysis have shown that overall it contains four main structural domains [2,4]. These domains are a variable N-terminal segment, a highly conserved core region, a variable spacer and a C-terminal conserved peptide. The functions of the N-terminal segment and spacer are not yet known. FtsZ protein in its pure form has been shown to possess GTPase activity. It binds and hydrolyses GTP. Self-assembly of FtsZ is a sequel to its GTP binding and subsequent GTPase activity [6,7]. The core region has the tubulin signature motif; GTP binding and hydrolysis are ascribed to this motif. The core region has two independently folding N-terminal and C-terminal segments. The N-terminal segment contains the GTP-binding site and binds the bottom portion of the adjacent monomer in the protofilament. On the other hand, the C-terminal segment binds the top portion of the adjacent monomer in the protofilament [2,6].

FtsZ undergoes self-assembly to form a circumferential dynamic Z-ring at the centre of the cell, and recruits approx. ten other proteins during progression and completion of bacterial cell division. Polymerization and GTP hydrolysis have been shown to be at the root of the dynamics of the Z-ring. In its absence, bacterial cell division gets inhibited, although DNA replication and nucleoid segregation could occur normally. Such a condition paves the way for a filamentous phenotype. The aberration eventually leads to cell lethality. In addition to cell division, FtsZ has been shown also to play a significant role in the division of organelles, such as plastids, mitochondria, etc. [2,3].

The current model for the role of FtsZ in cell division envisages that the FtsZ ring functions as the scaffold for the assembly of many factors required for the cell division. The constriction of the ring facilitates the separation of two daughter cells. This is a dynamic process, where the reversal of the assembly and subsequent formation of new FtsZ rings assemble at the midlines of the new daughter cells. In a recent study [8], it has been shown that ClpxX protein helps to disassemble the FtsZ ring following cell division. This implies that there is not a spontaneous reversibility; other proteins play a role in the process. The C-terminal part of the FtsZ protein has the important structural role of making contact with other proteins involved in cell division (such as ZipA, FtsA and FtsW). Its role is in the attachment of the Z-ring to the membrane with the help of these proteins. One unresolved question of importance in this regard is whether FtsZ protein acts as a scaffold for other proteins that generate the force, or whether the constriction force necessary is generated by the FtsZ protein itself [2]. Studies to answer this question will require diverse approaches, such as structural biological, biochemical, genomic and cell-imaging-based techniques. Recently, there has been a report modelling the physics of FtsZ assembly and force generation [9].

Apart from its central role in bacterial kinesis, another important feature of the FtsZ protein is that it is conserved in virtually all eubacteria, archaea and chloroplasts [1,2]. Therefore it has turned out to be a potential drug target. An additional factor contributing towards FtsZ protein as a drug target is that it is constitutively expressed, with its concentration remaining invariant during the cell cycle; therefore it is less likely that FtsZ might escape drug interaction. However, the prime necessity of a successful antibacterial drug is its specificity for the target and low host toxicity. Many antibacterial agents do not pass these stringent criteria. Any proposed agent targeting FtsZ protein should qualify as a drug, provided it meets the above criteria. On the negative side, the dose of the drug required to inhibit the bacterial kinesis by targeting FtsZ needs to be high, with the risk of potential toxicity to the host. Furthermore, in spite of the high level of homology among species for this protein, the drug-binding pockets across the species need not be equivalent. As a result, the drug might fail to be a broad-spectrum antibiotic.

From a general perspective, if we make an overview of FtsZ assembly in the Z-ring, any one or more of the following sequential steps could be a potential drug target: (1) division site selection and longitudinal association of FtsZ monomers; (2) lateral interactions between protofilaments; (3) Z-ring formation, recruitment of division proteins; and (4) constriction and cell division. Among these steps, the first one originates from the GTPase activity of FtsZ protein. In addition to FtsZ alone, interaction between FtsZ and cell division proteins is also an antibacterial drug target. One should not overlook the fact that cell division proteins are not conserved across the majority of bacteria, thereby limiting the broad spectrum of this class of compounds as drugs. A comprehensively written recent review article by Kapoor and Panda [10] has summarized the mode of actions of different drugs working via one or more of the above mechanisms. Table 1 enlists the natural compounds with anti-FtsZ activity. These compounds target one or more of the four steps mentioned earlier in the paragraph. It may be noted that a majority of the naturally occurring compounds with anti-FtsZ activity work at the level of inhibition of FtsZ polymerization via inhibition of its GTPase activity.

Table 1
Compound Source Anti-FtsZ activity (tested in) Mode of action on FtsZ 
Berberine Berberis aquifolium, Berberis aristate Escherichia coli Steps 1, 2 and 3 
Cinnamaldehyde Cinnamonium cassie E. coli Step 1 
Curcumin Curcurna longa B. subtilis Step 1 
Dichamentin Uvaria charnae − Step 1 
Sanguinarine Sanguinaria canadensis B. subtilis, E. coli Steps 1 and 2 
Totarol Podocarpus totatara B. subtilis, Mycobacterium Step 1 
Viridotoxin Aspergillus viridonutans Broad spectrum Step 1 
Compound Source Anti-FtsZ activity (tested in) Mode of action on FtsZ 
Berberine Berberis aquifolium, Berberis aristate Escherichia coli Steps 1, 2 and 3 
Cinnamaldehyde Cinnamonium cassie E. coli Step 1 
Curcumin Curcurna longa B. subtilis Step 1 
Dichamentin Uvaria charnae − Step 1 
Sanguinarine Sanguinaria canadensis B. subtilis, E. coli Steps 1 and 2 
Totarol Podocarpus totatara B. subtilis, Mycobacterium Step 1 
Viridotoxin Aspergillus viridonutans Broad spectrum Step 1 

Although the molecular basis of the inhibitory activity of these small molecules has been worked out, in many cases the detailed structure–activity relationships for these molecules are yet to be studied in depth. Another notable feature of some of this class of compounds, such as curcumin and sanguinarine, is that they also modulate different biochemical pathways in eukaryotes. Sanguinarine has been reported recently to induce epigenetic alterations via association with core histones in addition to chromosomal DNA [11]. There are also synthetic and semisynthetic molecules inhibiting FtsZ function. These molecules have been mostly developed on the basis of the skeleton structure of naturally occurring compounds. An in silico approach followed by high-throughput screening might go a long way towards developing this class of compounds.

In the latest issue of the Biochemical Journal, Beuria et al. [12] have reported a novel antibacterial agent, OTBA (3-5-[4-oxo-2thioxo-3-(3-trifluoromethylphenyl)-thiazolidin-5-ylidenemethyl]-furan-2-yl}-benzoic acid; structure shown in Figure 1), that targets the FtsZ assembly dynamics. They have used the novel approach of screening molecules with the potential to promote the assembly of FtsZ via association with the protein. As a consequence of this tight association, bundling of FtsZ protofilaments occurs. Dilution-induced disassembly of the protofilaments is thus inhibited. The net effect is the inhibition of bacterial cytokinesis, because both hyperstabilization and destabilization could in principle hinder the normal functioning of the Z-ring, leading to an inhibition of cell proliferation. The evidence presented in the paper indicates that, among a large number of compounds tested, OTBA singularly inhibits bacterial cell proliferation by the above mechanism that is different from that of the reported inhibitors of FtsZ assembly. The results also suggest that the dynamics of the Z-ring needs to be properly tuned for the appropriate orchestration of the bacterial division.

Chemical structure of OTBA

Owing to the similarities between FtsZ and tubulin, the first concern when identifying an FtsZ-targeted antibacterial agent is that it should not affect the microtubules of the host cells. OTBA inhibits bacterial cell proliferation with an IC50 of 1.1 μM, whereas it has no significant effect on microtubule organization in HeLa cells, even at a concentration 15 times higher than its IC50 in Bacillus subtilis cells. In addition, OTBA does not affect the polymerization of microtubule protein in vitro, suggesting that OTBA has a low toxicity on mammalian cells. Therefore this compound could be a potential lead molecule for the development of better and more effective analogues. In addition, the paper by Beuria et al. [12] has also thrown light on the molecular basis of the formation and functioning of the cytokinetic Z-ring.

One of the approaches utilized for cancer chemotherapy is to employ agents which arrest cell division via association with tubulin. FtsZ protein has a domain that is structurally analogous with that of tubulin; therefore any drug targeting the function of FtsZ protein can be compared with anticancer agents mentioned above. In their latest paper, Beuria et al. [12] have compared the activity of OTBA with the anticancer agent paclitaxil (taxol), which stands in the way of mammalian cell proliferation via stabilization of microtubules, thereby perturbing the mitotic spindle assembly.

In conclusion, the results presented by Beuria et al. [12] can be acclaimed as a significant step forward in presenting a new FtsZ-targeting antibacterial drug with a broad range of activity and higher level of selectivity associated with lower toxicity for the host.

Abbreviations

     
  • OTBA

    (3-{5-[4-oxo-2-thioxo-3-(3-trifluoromethylphenyl)-thiazolidin-5-ylidenemethyl]-furan-2-yl}-benzoic acid)

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