FtsZ consists of a conserved globular core (residues 10–316) comprising an N-terminal domain (H1–H6, S1–S6), a core helix (H7), a spacer loop (T7 loop) and a C-terminal domain (H8–H10, S7–S10), which in turn is connected to a conserved peptide at the extreme C terminus (residues 369–383) by a flexible unstructured linker (317–368) (de Boer et al., 1992; Löwe and Amos, 1998; Nogales et al., 1998b) (Figure 1). In some species, such as the archaeon Methanocaldococcus jannaschii, FtsZ contains an additional helix (H0) in its N-terminal subdomain (Löwe, 1998).

In E. coli FtsZ, the N-terminal domain spans residues 1–177. It contains an unstructured and poorly conserved extreme N terminus and a highly conserved NBD (Löwe and Amos, 1998; Vaughan et al., 2004; Gardner et al., 2013). Although the N-terminal domain contains the NDB, it is not sufficient for hydrolyzing GTP (Jindal and Panda, 2013). In E. coli, H7 extends from residues 178 to 201, connects the N-terminal and C-terminal domains of the core, and acts like a sliding door for the opening and closing of the IDC. Some residues of H7 are crucial for FtsZ assembly. For example, a single mutation in Bacillus subtilis FtsZ (BsFtsZ) R191 can impede FtsZ assembly (Dhaked et al., 2016).

The highly conserved T7 loop in E. coli FtsZ (residues 202 – 209) connects H7 to H8 of the C-terminal subdomain and contains a conserved GXXNXD sequence that is important for GTP hydrolysis. Upon FtsZ assembly, the T7 loop of one FtsZ monomer inserts into the GTP binding pocket of the adjacent FtsZ monomer and initiates GTP hydrolysis (Löwe and Amos, 1999; Scheffers et al., 2002). Drug molecules that bind to this site affect GTPase activity of FtsZ.

The C-terminal domain of the globular core of FtsZ (residues 210 to 316) is highly conserved both in sequence and structure. It consists of helices H8–H10 and beta sheets S7–S10. H10 is notably rich in acidic residues and interacts with Min proteins (Taviti and Beuria, 2017). This domain is followed by an unstructured C-terminal linker (CTL) that is highly variable both in composition and length (Taviti and Beuria, 2017). In E. coli, the CTL is ∼ 52 residues (317 – 369). In most FtsZs, the CTL connects the globular core domain of FtsZ with a highly conserved 10–20 residue peptide at the extreme end of the C-terminus called the C-terminal peptide (CTP) (Cohan et al., 2020). Although this peptide (residues 369–379 in E. coli) is not required for FtsZ assembly, it is crucial for interactions with other membrane-associated cell division proteins like ZipA and FtsA (Ma and Margolin, 1999; Ortiz et al., 2016). Residues D373, I374, F377 and L378 of E. coli FtsZ are specifically involved in these protein-protein interactions (Buske and Levin, 2012). As a result, deletion of the CTP blocks FtsZ functions and bacterial division (Din et al., 1998).

FtsZ, in the presence of GTP, polymerizes into head-to-tail protofilaments (Bramhill and Thompson, 1994; Mukherjee and Lutkenhaus, 1994), which then coalesce to form the Z-ring (Haeusser and Margolin, 2016). The Z–ring is anchored to the membrane with the help of other essential cell division proteins, such as FtsA and ZipA of E. coli (Pichoff and Lutkenhaus, 2002). In Gram positive bacteria as well as FtsZ-containing archaea, SepF is the key membrane anchor for FtsZ (Duman et al., 2013; Nussbaum et al., 2021; White and Eswara, 2021).

In vitro studies suggest that FtsZ assembles into short protofilaments made up of ∼30 subunits that combine laterally to form the Z-ring (Erickson et al., 2010). These lateral interactions between FtsZ protofilaments help to drive division septum formation (Krupka and Margolin, 2018; Squyres et al., 2021; Whitley et al., 2021). A single FtsZ protofilament is ∼5 nm thick with slightly curved morphology, which becomes highly curved upon GTP hydrolysis (Lu et al., 2000; Romberg et al., 2001). One model proposed that GTP hydrolysis provides the required force for Z-ring constriction and septation (Allard and Cytrynbaum, 2009). As GTP hydrolysis is induced upon FtsZ assembly into polymers, FtsZ subunits within the Z ring are highly dynamic, with a half time of FtsZ subunit turnover as low as 8–9 s in E. coli and B. subtilis (Anderson et al., 2004). This turnover results from treadmilling, which allows FtsZ polymers to travel circumferentially around the site of septum formation by loss of subunits at one polymer end and gain of subunits at the other (Bisson-Filho et al., 2017; Yang et al., 2017). Surprisingly, only about ∼30% of the FtsZ in E. coli cells is actually in the Z-ring at any one time, while the remaining FtsZ circulates in a cytoplasmic pool that is continuously exchanged with treadmilling FtsZ polymers that comprise the Z ring (Stricker et al., 2002). Despite the rapid turnover observed in cells, purified FtsZ in solution hydrolyzes its bound GTP at a rate of only ∼2 GTP per FtsZ molecule per minute (Lu et al., 1998), suggesting that cellular factors may enhance FtsZ GTPase activity. Molecular dynamics simulations of FtsZ dimers predict the forces generated by GTP hydrolysis to be ∼30 pN per FtsZ monomer, which is within the range of force required (8 – 80 pN) to drive cytokinesis as mentioned above (Lan et al., 2007; Hsin et al., 2012). Nonetheless, inward growth of the cell division septum likely contributes significantly to the constriction of the Z ring.

As mentioned above, there are two main binding pockets for drug binding to FtsZ, the NBD and IDC (Figure 2). The NBD, at the interface between FtsZ monomers, includes helices H1–H6, sheets S1–S6 and the T1–T6 loop. It also includes the N-terminal part of H7 (Löwe, 1998; Panda et al., 2016). Seven distinct regions in FtsZ interact with GTP. The T1 loop interacts with the phosphate and the guanine base, whereas the T2 & T3 loops interact with the β- and γ-phosphates (Nogales et al., 1998a; Panda et al., 2016). The T4 loop contains the tubulin signature motif, which interacts with the α and β- phosphates (Mukherjee et al., 1993; de Pereda et al., 1996). The T5-loop interacts with the ribose sugar. The T6 loop possesses an asparagine residue (N165 in E. coli), which interacts with the guanine base via a hydrogen bond and is conserved in both FtsZ and tubulin (Panda et al., 2016). The guanine base is mainly recognized by amino acid residues present within the H7 helix. There are several small molecules that are known to interact with the FtsZ NBD (Table 1). Most of these could either interact with microtubules or were screened from a library of microtubule-interacting molecules. As the NBD is highly conserved in both FtsZ and tubulin, any molecules that bind to it may be toxic to mammalian cells. Indeed, curcumin, which binds the NBD of both FtsZ and tubulin, has an MIC of 100 μM in B. subtilis and an IC₅₀ of 18 μM for HeLa cells (Rai et al., 2008; Chakraborti et al., 2011). Some other examples of this cross-toxicity are summarized in Table 1 (IC₅₀/MIC).

The second major binding site in FtsZ that interacts with small molecules is the IDC, formed by the C-terminal half of the H7 helix, the T7 loop and the beta sheets in the C-terminal domain (Figure 1C) (Sun et al., 2014). The size of the cleft, the number of amino acid residues, their conservation and types vary among different bacterial species (Figure 3 and Table 2). For example, the IDC is less conserved in Gram negative bacteria (fewer than nine conserved residues) than Gram positive bacteria (more than nine conserved residues). The size of the IDC also varies among bacterial species, depending on the curvature of the H7 helix. For example, in the GTP bound state of Staphylococcus aureus FtsZ the curvature of the H7 helix decreases, which in turn increases the size of the cleft opening. The T7 loop of FtsZ also influences the cleft opening size. In bacteria such as S. aureus and B. subtilis, the T7 loop in the GTP bound state shifts downward, resulting in a larger cleft opening compared to the GDP bound state (Kusuma et al., 2019). As the T7 loop of one FtsZ subunit is inserted into the nucleotide-binding pocket of the adjacent FtsZ subunit to trigger GTPase activity (Panda et al., 2016), this loop is crucial for modulating FtsZ treadmilling dynamics.

In most bacterial species, many archaea, some chloroplasts and a few primitive mitochondria, the globular domains of FtsZ share 40–50% structural and functional similarity (Erickson, 1998; Tripathy and Sahu, 2019). Compared to the NBD, which exhibits 48–67% sequence identity among FtsZs from E. coli, Pseudomonas aeruginosa, B. subtilis, S. aureus, Mycobacterium tuberculosis, Aquifex aeolicus, and the archaeon M. jannaschii, the IDC shows less sequence conservation (34–59% identity) (Casiraghi et al., 2020). The three-dimensional structure of the NBD is quite similar among FtsZs from different bacterial species, whereas it is slightly different among various IDCs because of how the C-terminal beta sheets are organized (Kusuma et al., 2019).

The sequence level as well as structural level variability of IDCs among different FtsZs should facilitate the design of species-specific antibiotics (Casiraghi et al., 2020). For example, PC190723, an IDC inhibitor that will be described in greater detail below, is most effective against organisms that have a valine at the equivalent position of 307 in E. coli FtsZ, such as S. aureus and B. subtilis. In contrast, PC190723 is ineffective toward E. coli (Haydon et al., 2008), although inactivating the AcrAB efflux pump of E. coli significantly enhances susceptibility to the PC190723 prodrug TXY436, suggesting that resistance of Gram negative species to this compound is in part due its rapid clearance from the cytoplasm (Kaul et al., 2014).

Although the sequence and structure of the IDC as a whole is not highly conserved, components of the IDC, like the T7 loop, are highly conserved (de Pereda et al., 1996). Kusuma et al. compared the tertiary structure of staphylococcal and non-staphylococcal FtsZ proteins and showed that their structures differ mainly because of variations in the curvature of the H7-helix and organization of the C-terminal β-sheet (Kusuma et al., 2019). Superimposition of staphylococcal and non-staphylococcal FtsZs revealed that staphylococcal FtsZs were similar to each other, with an RMSD value of 0.3 Å, and non-staphylococcal FtsZs were more structurally variable among themselves (RMSD ∼1.3 Å). Conversely, S. aureus FtsZ (hereafter referred to as SaFtsZ) showed much higher variation (RMSD ∼3 Å) when superimposed onto non-staphylococcal FtsZ. This difference was mainly due to the diversity in the arrangement of C-terminal β- sheets. Similarly, when compared the drug binding sites, both staphylococcal and non-staphylococcal FtsZ showed no significant structural differences in their GTP binding sites and T7 loops, whereas their IDCs were quite divergent. As the cleft opening size of the IDC and the curvature of helix H7 vary significantly between staphylococcal and non-staphylococcal FtsZs, compounds that bind to a staphylococcal FtsZ may not bind to non-staphylococcal FtsZ with similar affinity. Likewise, a compound targeting a non-staphylococcal FtsZ may not bind to other non-staphylococcal FtsZs with the same affinity (Kusuma et al., 2019).

Microtubules, formed by polymerization of α-β- tubulin, are eukaryotic cytoskeletal proteins that play important roles in several cellular processes such as cell division, cell motility, intracellular transport and maintaining cell shape. Along with the GTP binding pocket, microtubules contain at least four major drug binding sites, including those for vinblastine, colchicine, laulimalide, and taxane (Figure 2A) (Lu et al., 2012). Vinblastine binds at the interface of the α-β- tubulin heterodimer (Figure 2B), which comprises the T7- loop, H10 and S9 strand of α tubulin and H6, the T5 loop and H6–H7 loop of β- tubulin (Steinmetz and Prota, 2018). The colchicine binding site includes the T7-loop, helices H7 and H8, and strands S8 and S9 of β- tubulin plus the T5 loop of α tubulin (Ravelli et al., 2004). The laulimalide binding site comprises helices H9 and H10 and the H10-S9 loop of β- tubulin, whereas taxol, one of three taxane derivatives commonly used as an anticancer drug, binds to the β- tubulin H7, S7, H6–H7 loop, S7–H9 (M-loop) and S9–S10 loop (Prota et al., 2014; Kellogg et al., 2017).

In contrast to this diversity of binding sites in microtubules, only the NBD and IDC of FtsZ have been identified as drug binding targets. The IDC of FtsZ consists mainly of the H7 helix, S7–S10 strands and T7 loop that structurally map to the taxol and colchicine binding sites in tubulin (Table 3 and Figure 2). Because the IDC has a lower level of sequence and structural similarity with tubulin compared with the NBD, colchicine and taxane can interact with FtsZ, but the interacting residues as well as binding affinity do not match with tubulin (White et al., 2002; Haydon et al., 2008). For example, although colchicine can bind to the IDC in FtsZ, the colchicine binding pocket of tubulin has no sequence similarity with the colchicine binding site in FtsZ from M. tuberculosis (White et al., 2002; Mathew et al., 2016). In another example, PC190723 (an IDC-specific inhibitor) binds to the taxol site on tubulin, but was >64-times more inhibitory to FtsZ than to tubulin (Haydon et al., 2008). Similarly, SB-RA-2001, a taxane derivative, binds to the IDC of BsFtsZ, but when the binding site was superimposed onto the taxol site of tubulin, no identical residues were found; it also binds only very weakly to tubulin (Singh et al., 2014). Consequently, the drugs that bind to the IDC in FtsZ do not interact with tubulin with similar affinity and thus are less likely to be toxic to mammalian cells.

The N-terminal domains of FtsZ share high sequence identity in both Gram-positive bacteria (56–89%) and Gram-negative bacteria (43–84%), whereas lower sequence identities (30–70%) are shared within the C-terminal domains and IDCs. Inter-domain clefts of diverse FtsZs are composed of mostly hydrophobic residues along with a few polar and charged amino acids. Available crystallographic structures for protein-ligand interactions indicate that most small molecules prefer to bind to the hydrophobic pockets of their protein targets (Guo et al., 2015). Thus, the hydrophobic residues in the FtsZ IDC likely enhance the binding of organic molecules in aqueous environments, making the IDC a better target for small molecule inhibitors.

Some residues within the IDC are widely conserved in all bacterial species, some are conserved only among the Gram positive bacteria or in Gram negative bacteria, whereas other residues are specific to a particular species. For example, residues V189, Q192, G193, Q195, G196, I197, D199, L200, I201, V203, S204, G205, E206, V207, N208, L209, D210, M226, G227, I228, L261, M262, N263, T265, G295, T296, V297, T309, V310, and I311 are located within 6 Å of the IDC of SaFtsZ. The corresponding residues in EcFtsZ are shown in Table 4. Of these, N208, D210, G227, and G295 are conserved throughout bacteria that have FtsZ; G193, Q195, I197, L200, I201, M226, T309, and V310 are conserved mostly in Gram-positive species, and the remaining residues are not conserved (Figure 3C). These residues are involved in formation of different bonds with the small molecules–hydrogen bonds, hydrophobic, van der Waals, amide bonds or other types of interactions–and depend upon the chemical nature of the inhibitors and the interacting residues. For example, V207 and N263 are mainly involved in hydrogen bonding with PC190723, while L200 and I311 form hydrophobic interactions (Matsui et al., 2012). The size of the IDC, the number of amino acid residues and their types vary among different bacterial species. For example, a multiple sequence alignment of IDCs from 12 bacteria showed that 6–12 residues are conserved between Gram-positive and Gram-negative species, whereas more than 12 residues are conserved when aligned among only Gram positive bacteria.

Although the IDC mostly consists of hydrophobic residues, it also contains several conserved hydrophilic residues that are important for interaction with small molecule inhibitors. Similarly, many residues are important for interaction with multiple inhibitors. For example, residues in EcFtsZ such as G191, G195, L199, M206, N207, V208, N263, S297, R307 and T309 and their equivalent residues in both B. subtilis and S. aureus (Q192, G196, L200, V207, N208, L209, N263, V297, V307, and T309 in the latter species) mostly interact with more than one FtsZ inhibitor (Figure 4A and Table 2). Our analysis of the published literature suggests that most FtsZ inhibitors that target the IDC have higher IC₅₀/MIC ratios than inhibitors that target the NBD (Figure 4B). Although IDCs from different bacteria are highly hydrophobic, their lower sequence conservation and variable cleft openings should potentially facilitate development of species-specific antibacterial agents.

Structural organization of the IDC indicates that the cleft has a specific size and a specific cleft opening. The cleft opening changes for different conformers of FtsZ such as GDP/GTP bound forms, and monomeric/polymeric FtsZ. Further, the size of the cleft opening differs in different bacterial species. Recent analysis of crystal structures of diverse FtsZs by Kusuma et al. indicated that IDC size depends upon the curvature of H7: if its curvature increases, the size of the IDC opening decreases, and vice-versa (Figure 5) (Kusuma et al., 2019). Using in silico analysis, they further measured the cleft opening size and the curvature of the H7 helix in Staphylococcus and non-Staphylococcus FtsZs, providing clues to the molecular accessibility of IDCs from different bacterial species (Kusuma et al., 2019). Their analysis shows that the curvature of H7 in SaFtsZ is 140.3° (PDB ID: 3WGN) and the cleft opening size is 15.9 Å, whereas, in BsFtsZ it is 164.5° (PDB ID: 2RHO) and 7.5 Å, respectively. The same study showed that the drug-binding pocket is also subject to species-level variations: S. aureus has the widest cleft opening (∼15 Å), whereas in B. subtilis, M. tuberculosis, A. aeolicus, and P. aeruginosa the cleft opening size is 9–10 Å. The IDC binder PC190723 has a size of 14.1 Å, with higher affinity for SaFtsZ than BsFtsZ. This wider cleft opening of SaFtsZ vs. BsFtsZ probably facilitates PC190723 entry and binding, and may be the major reason why it inhibits S. aureus cell division more effectively than that of B. subtilis.

Similarly, molecular dynamic simulations indicate that in the GTP bound state, the H7 helix is twisted backward and the T7 loop shifts downward, opening the cleft, whereas in GDP bound FtsZ the H7-helix is relaxed and the T7 loop shifts upward, closing the cleft’s opening. The size of the cleft opening in GDP and GTP bound SaFtsZ varies between 15 and 20 Å (Kusuma et al., 2019). This model is supported by fluorescence anisotropy experiments showing that a fluorescent analog of PC190723, a nitrobenzoxadiazole probe, specifically binds to the polymeric form of FtsZ (Artola et al., 2017). Molecular dynamics simulations suggest that in monomeric FtsZ the cleft is in a closed or relaxed conformation, preventing the probe from interacting with FtsZ, whereas in polymeric FtsZ the cleft is in the open or tense conformation (Wagstaff et al., 2017; Schumacher et al., 2020), allowing interaction with FtsZ and resulting in fluorescence. In silico analysis of the PC190723 binding pocket in the IDC in different bacterial species showed that the microenvironment of the binding pocket affects the drug’s affinity toward FtsZ (Miguel et al., 2015). Analysis of FtsZ crystal structures and molecular dynamics trajectories showed that the conformation of the PC190723 binding pocket depends upon multiple factors such as bacterial species, genetic alterations, allosteric binding and polymerization state (Miguel et al., 2015). In particular, FtsZ polymerization and allosteric binding of the guanosine nucleotide may play a crucial role in stabilizing the PC190723 pocket. For example, for PC190723, the GDP-bound SaFtsZ has a pocket score of -10.75 (PDB ID: 3VO8), whereas FtsZ without nucleotide has a pocket score of -4.29 (PDB ID 3VO9). Similarly, amino acid substitutions G193D, G196C, and N263K in SaFtsZ change the microenvironment of the binding pocket, significantly affecting the binding of PC190723 and leading to drug resistance (Haydon et al., 2008; Miguel et al., 2015).

Ohashi et al. originally showed that a benzamide derivative, 3-MBA (3-Methoxybenzamide), inhibits the proliferation of bacteria by targeting FtsZ (Ohashi et al., 1999). Although 3-MBA has low antibacterial activity (MIC = 2048 μg/mL), it provided a strong starting point for FtsZ-targeted fragment-based drug discovery. Screening more than 500 benzamide analogs led to the discovery of the aforementioned PC190723, which contains a thiazolopyridine moiety fused to the benzamide by an ether linkage that makes it ∼2000 times more potent than the parent 3-MBA (MIC = 0.5–1 μg/ml). Molecular docking and X-ray crystallography demonstrated that PC190723 binds to the IDC, interacting specifically with R191, Q192, N263, V307, and T309 in BsFtsZ (Haydon et al., 2008). Similarly, crystallography showed that PC190723 binding site in SaFtsZ comprises Q192, G193,G196, I197, D199, L200, V203, G205, V207, N208, L209, M226, G227, I228, N263, G295, T296, T309, V310, and I311 (Table 4) (Matsui et al., 2012). PC190723 binding to the IDC disrupts the normal assembly of the Z ring by causing multiple FtsZ aggregates to distribute throughout the cell that are not able to form a coherent Z ring (Haydon et al., 2008).

Not surprisingly, S. aureus developed resistance to PC190723 by altering SaFtsZ residues R191 G193, G196, V214, N263, or G266. G196 mainly interacts with the thiazolopyridine moiety and changes at this residue are commonly found in PC190723- resistant S. aureus. Interestingly, however, some PC190723-resistant mutants such as R191P and G196A in SaFtsZ and G196S in BsFtsZ remain sensitive to 3-MBA, suggesting that the less specific 3-MBA can still bind to an IDC pocket that occludes PC190723 binding (Adams et al., 2016). Moreover, some substitutions in FtsZ that render cells non-susceptible to PC190723, such as R191P, G193D, and G266S, at the same time confer benzamide dependence for normal cell division (Adams et al., 2016), although the mechanism for this drug dependent function of FtsZ is not clear. Interestingly, a 3-MBA-resistant mutant (A47) remains susceptible to PC190723 (Haydon et al., 2008).

A notable advance in optimizing benzamide action against FtsZ was the replacement of the chlorine atom on the pyridyl ring of PC190723 with a CF3 group, exemplified by a derivative called TXA707, which increases the drug’s metabolic stability and anti-staphylococcal activity (MIC: 0.25–2 μg/ml). Nonetheless, TXA707 is less effective on FtsZs with residue changes at G196 and others that mediate resistance, probably for the reason discussed above. A modification made in TXA707, in which a five membered oxazole and six membered phenyl ring (i.e., TXA6101) are flexibly linked, not only improved the binding affinity but also increased its activity against both wild-type methicillin resistant S. aureus (MRSA) and mutants carrying residue changes at FtsZ G196 (MIC = 0.125 μg/mL and 1 μg/mL, respectively) (Fujita et al., 2017). Crystallography and biochemical studies showed that both TXA707 and TXA6101 interact with the IDC. TXA6101 induces a conformational rearrangement of I197, M226, and I311 that leads to the formation of an inner hydrophobic pocket, with M226 acting as a gate that opens access to the pocket (Fujita et al., 2017).

Further advances have been made using improved benzamide prodrugs. TXY541 is a prodrug of PC190723 that is 143-times more soluble in an aqueous acid vehicle than PC190723 (Kaul et al., 2013). A prodrug of TXA707, TXA709, is structurally similar to TXY541 except that TXA709 contains a CF3 group instead of Cl group on the pyridyl ring, which increases the metabolic stability of the compound. Currently, TXA709 is in phase-I clinical trials. Recent reports showed that clinically isolated MRSA display resistance toward TXA709 at a frequency of 1 x 10^–8, which is similar to that for PC190723. TXA709-resistant isolates carried mutations in FtsZ at G196S, N263K, G193D, G196C, and G196A, similar to residues that confer S. aureus resistance to PC190723 (Kaul et al., 2015).

In another study of benzamides, Bi et al. designed and synthesized a series of 3-substituted 2,6-difluorobenzamide derivatives, of which a chloroalkoxy derivative (7), a 3-bromoalkoxy derivative (12) and a 3-alkyloxy derivative (17) exhibited good antibacterial activity against B. subtilis and susceptible/resistant S. aureus (Bi et al., 2017). Using structure-based drug design to target the IDC, they designed and synthesized a series of isoxazole (isoxazol-3-yl- and isoxazol-5-yl) containing benzamide derivatives. Some of these isoxazol-5-yl benzamide derivatives (B14) were ∼32 fold more potent against B. subtilis than PC190723 (Bi et al., 2018). In another study, Bi et al synthesized a series of 1, 3, 4-oxadiazol-2-one containing benzamide derivatives. Out of many derivatives, compound A14 showed the highest antibacterial activity against Gram positive bacteria (MIC 0.125-1 μg/mL) and less cytotoxicity against HeLa cells (IC₅₀ > 64 μg/mL). In silico docking revealed that compound A14 binds to the IDC (Bi et al., 2019). A recent study of benzodioxane-benzamides identified a derivative (compound 8) with very high potency against MRSA and B. subtilis, with MICs at or below 0.1 μg/ml, good solubility, and very low toxicity to human cells. Like the original PC190723 (Haydon et al., 2008), compound 8 caused the delocalization of Z rings in B. subtilis into subcellular foci that were unable to function in cell division (Straniero et al., 2021) (Figure 6).

Using cell-based screening, Lui et al screened 47 derivatives of 3-aminobenzamide and showed that their compound 28 interacts with the FtsZ IDC. This compound exhibits high antibacterial activity (MIC 0.5–1 μg/mL against S. aureus) and less cytotoxicity (IC₅₀ ≥ 100 μM, mouse L929 cell line) and worked in synergy with β-lactam antibiotics. Molecular docking studies showed that the C3 amino group of compound 28 interacts with the hydroxyl group of T309 in SaFtsZ. The other SaFtsZ residues in its proximity are G193, G196, M262, and N263 (Table 4). They also found that the M262I residue change is resistant to this molecule (Lui et al., 2019).

Recently, Ferrer-González et al. developed a structure-guided fluorescent benzamide derivative by conjugating BODIPY to an oxazole benzamide FtsZ inhibitor (BOFP). BOFP binds to FtsZ from both Gram positive and Gram negative bacteria with K_ds of 0.6–4.6 and 0.2–0.8 μM, respectively (Ferrer-Gonzalez et al., 2019). BOFP binds to the IDC, where the BODIPY moiety interacts with residues I228 and V307 of BsFtsZ (Table 4). As it can label FtsZs within diverse bacteria, BOFP holds great promise for the screening of non-fluorescent FtsZ inhibitors and determining whether they perturb Z ring assembly in cells. In a recent study, Huecas et al. developed a competitive binding assay using specific high-affinity fluorescent probes to screen allosteric compounds that can interact with the IDC and inhibit FtsZ assembly. The probes displayed higher anisotropy in the presence of FtsZ polymer, where the IDC is open, compared with FtsZ monomers, where the IDC is closed. The specificity of this probe was assessed using a competitive assay with PC190723. The study demonstrated that the anisotropy of the probe decreased considerably upon binding of the IDC specific inhibitor PC190723, whereas it did not change upon binding of non-specific inhibitors. Thus, this probe can be used to identify inhibitors that specifically bind to the IDC (Artola et al., 2017; Huecas et al., 2021).

Berberine is a benzylisoquinoline alkaloid that has been used as an antimicrobial therapeutic for centuries. After identification of its interaction with FtsZ, Wong et al. used in silico structure-based design to synthesize a number of 9-phenoxyalkyl berberine derivatives that bind to the IDC of SaFtsZ (Sun et al., 2014). A positively charged amine on these derivatives interacts with SaFtsZ residue D199 and a C-9 methoxy binds to several hydrophobic residues (I228, V230 and V307) in the IDC. Modifications of these two moieties make the derivatives more potent than berberine (MIC = 100–400 μg/mL) and enable them to inhibit growth of MRSA and vancomycin-resistant Enterococci (VRE), with MICs of 2–8 μg/mL and 4–16 μg/mL, respectively. These compounds also exhibit moderate antimicrobial activity against Gram negative strains such as E. coli (MIC 32–128 μg/mL). Similarly, 9-phenoxy berberine derivatives inhibit FtsZ GTPase activity (IC₅₀ 37.8 – 63.7 μM) more potently than the parent berberine molecule (IC₅₀ = 272 μM) and showed similar effects on FtsZ polymerization. This confirms that the substitution of the 9-phenoxy group in berberine increases its affinity toward FtsZ and its antibacterial activity.

Quinolines fused with a thiazole orange derivative confer broad spectrum antibacterial activities. Among them, 2-((E)-4- Hydroxystyryl)-1-methyl-4-((Z)-(3-methylbenzo[d]thiazol-2 (3H)-ylidene) methyl) quinolin-1-ium iodide (1) (compound-1) exhibits high antibacterial activity against S. aureus (MIC ∼ 1.5–3 μg/mL), other staphylococci (MIC ∼ 0.75–3.0 μg/mL) and E. coli (MIC ∼ 1.5 μg/mL). This compound enhances FtsZ bundling at lower concentrations (10 nm – 90 nm), inhibits GTPase activity (IC₅₀ = 5 μg/mL) and is significantly less toxic to mammalian cells (IC₅₀ = 98.15 μM). Molecular docking studies showed that it binds to the IDC of SaFtsZ through both hydrophobic interactions at residues L200, M226, I228, L261, V297, L302, V307, and I311 and hydrogen bonding to V203 and L209 (Table 4) (Sun et al., 2017b).

High throughput phenotypic screening by the NIH screened 215,110 molecules against the M. tuberculosis (Mtb) H37Rv strain and the data are freely available. Using the results from this screen, Mathew and coworkers found that quinoline and quinazoline can inhibit MtbFtsZ functions (Mathew et al., 2013). Subsequently, Cai et al. synthesized a series of 1-methylquinolinium derivatives (c1-c15) by combining an indole fragment at the 2-position with different amino groups at the 4-position (Cai et al., 2019). These compounds strongly inhibited FtsZ activity and growth of MRSA and VRE, with MIC values between 1 and 4 μg/mL. C2 and c9 derivatives enhanced bactericidal activity with an MIC of 1 μg/mL in S. aureus (ATCC 29213). Both compounds possess a common piperidine group at the 4-position of the 1-methylquinolinium core that might increase its antibacterial properties compared with other indole-quinolinium derivatives. Molecular docking studies predicted that these derivatives bind to the IDC of SaFtsZ mostly through hydrophobic interactions with residues Q192, G196, L200, V203, L209, M226, G227, I228, and V297 and electrostatic interaction with D199 (Table 4).

Several quinolone and quinoline derivatives exhibited antibacterial activity against Gram positive and Gram negative bacteria (Piddock and Walters, 1992; Aldred et al., 2014; Zhang et al., 2018). Sun et al. synthesized a series of quinoline derivatives containing the unique quaternary pyridinium core, many of which demonstrated antibacterial activities (Sun et al., 2017a). These compounds interacted hydrophobically with FtsZ residues D199, L200, V297, and V307, and the imino group of these derivatives could hydrogen-bond with FtsZ T309. These compounds showed ∼50-fold better antibacterial activity against B. subtilis (MIC ∼2–8 μg/mL) compared with berberine (MIC = 128 μg/mL). In their next study, Sun et al. synthesized sixteen 3-methylbenzo[d]thaizol-methylquinolinium derivatives with different groups added to the ortho-position of the 1-methylquinolinium core (Sun et al., 2018). One of the derivatives, A2, showed strong antibacterial activity (MIC = 1.5 μg/mL) by inhibiting FtsZ functions, and like the others, exhibited low toxicity toward mammalian cells (IC50 = 78.25 μM) (Sun et al., 2018). A docking study showed that A2 interacts with residues D199, L200, M226, I228, V297, T309, and I311 of SaFtsZ (Sun et al., 2018).

From previous studies, it was clear that thiazole and quinolinium groups are important for antibacterial activity. Li et al. synthesized various thiazole-quinolinium derivatives and evaluated their antibacterial activities against Gram positive and Gram negative species (Li et al., 2015). All compounds showed good antibacterial activity (MIC 1–32 μg/mL) against S. aureus. A methyl group substitution at the quinolinium ring resulted in better antibacterial potency than the bulky indolyl group. These derivatives were effective against antibiotic resistant strains, did not induce antibiotic resistance, and showed less cytotoxicity toward mammalian cells (16HBE, HK-2 L929 with IC50 12–26 μg/mL). Molecular docking studies determined that thiazole-quinolinium derivatives interact with the IDC through numerous hydrophobic bonds with residues D199, L200, M226, I228, V297 and van der Waals interactions with Q195, V310, G205, and I311 of SaFtsZ (Table 4).

Plumbagin (5-hydroxy-2-methyl-4, 4-naphthoquinone) is a naturally occurring naphthoquinone originally isolated from the plumbago plant (de Paiva et al., 2003; Aziz et al., 2008). It inhibits proliferation of diverse species such as S. aureus, P. aeruginosa, B. subtilis (MIC∼29 μM), Proteus vulgaris and M. smegmatis (MIC ∼ 31 μM) (de Paiva et al., 2003; Mathew et al., 2010). Plumbagin binds to BsFtsZ and inhibits its assembly and GTPase activity (Bhattacharya et al., 2013). In vitro and in silico assessment studies demonstrated that the plumbagin binding site is distinct from the NBD in BsFtsZ (Bhattacharya et al., 2013). The residues of BsFtsZ that constitute the plumbagin binding site include the H7 helix and other residues in the IDC such as R191, Q192, Q195, G196, D199, N263, T265, N299, V307, and T309 (Table 4). Of these, R191, Q195, D199, and N299 of BsFtsZ are involved in hydrogen bonding with plumbagin (Bhattacharya et al., 2013). Mutational studies confirmed that D199 and V307 of BsFtsZ play an important role in plumbagin binding. In silico studies showed that the plumbagin binding site in EcFtsZ includes residues G21, M104, T132, P134, E138, R142, N165, F182, A185, and L189 (Bhattacharya et al., 2013). Notably, these residues are in a completely different part of FtsZ than the plumbagin binding pocket of BsFtsZ, suggesting that FtsZs of different bacteria may have different ligand binding properties.

Since the discovery of penicillin, it is well known that bioactive molecules of fungi show strong antimicrobial properties. While screening 58 fungal compounds from 24 different genera, Wu et al. found that Penicillium cataractum SYPF 7131 has strong antibacterial activity against S. aureus (Wu et al., 2018). Out of the 8 known and unknown isolates from its fermentation broth, 3 compounds showed effective antibacterial activity (MIC 10–65 μg/mL) and strong interaction with FtsZ. An in silico study suggested that these compounds interact with the IDC of SaFtsZ by hydrogen bonding with residues G205, N263, T309, L209, G196, G227, and G193, and hydrophobic interaction with L200, L209, I311 L261, V307, V203, I228, I311 V297, and V203 (Table 4).

Doxorubicin is an anthracycline antibiotic which inhibits bacterial proliferation with moderate inhibition against E. coli (MIC 40 μM) and strong inhibition against S. aureus (MBC 5 μM). In the presence of doxorubicin, E. coli becomes highly filamentous without affecting chromosome segregation, indicating a cell division defect. Panda et al. showed that doxorubicin binds to a site in FtsZ distinct from the NBD. The amino sugar region of doxorubicin sits in a polar cavity and involves hydrogen-bond interactions with E32, R33, and D187 of EcFtsZ, whereas the ethyloxy side chain involves hydrogen-bond interaction with E305 (Panda et al., 2015). The hydrophobic part of the molecule (the aromatic rings) sits in a cavity lined by hydrophobic residues of FtsZ, e.g., V171, V188, M225, V229, and L248. The binding site contains many highly conserved residues, including E32, R33, V171, D187, V188, M225, G226, P247, L248, M302, N303, E305, and R307.

Rhodanine derivatives can perturb the assembly of FtsZ polymers and inhibit bacterial proliferation. Singh et al. screened a library of 151 rhodanine derivatives, of which 8 compounds showed good antibacterial activity (MIC ∼2 μM) and 3 specifically inhibited division of B. subtilis cells (Singh et al., 2012). One of these molecules, CCR-11, interacts with FtsZ with a Kd of 1.5 ± 0.3 μM and inhibits FtsZ assembly and GTPase activity. Docking studies revealed that CCR-11 binds to the IDC. The fluorine atoms of the CCR-11 trifluoromethylphenyl side chain interact with BsFtsZ T203 and CCR-11 also interacts with BsFtsZ G205, I207, L272, V275, and I298 through hydrophobic interactions. The thiazolidine ring of CCR-11 interacts with T203 and D199 of BsFtsZ. CCR-11 inhibited HeLa cell proliferation with an IC₅₀ value of 18.1 ± 0.2 μM, which is 6 times higher than the MIC (3 μM) of CCR11 on B. subtilis (Singh et al., 2012).

Ray et al. screened 100 benzamidazole compounds for them) ability to elongate B. subtilis cells, out of which one compound, N-(4-sec-butylphenyl)-2- (thiophen-2-yl)-1H-benzo[d]imidazole-4-carboxamide (BT-benzo-29), causes cell filamentation. BT-benzo-29 inhibits FtsZ functions by interacting with FtsZ (Kd = 24 ± 3 μM) and inhibits proliferation of B. subtilis with an MIC of 17 μM (Ray et al., 2015). A molecular docking study proposed that BT-benzo-29 binds to the BsFtsZ C-terminal portion of the globular domain, near the T7 loop. The interaction involves hydrogen bonding with L206 and S296 and hydrophobic interactions with D199, T203, P204, G205, L206, N208, L270, S271, L272, V275, S296, V297, I298, and E300 residues (Ray et al., 2015). Mutational studies showed that L272A and V275A mutants had weaker inhibitory effects on the assembly and GTPase activity. Unfortunately, BT-benzo-29 inhibits HeLa cell proliferation with an IC₅₀ 17 ± 2 μM, only ∼4 times higher than the IC₅₀ for B. subtilis.

Tiplaxtinin is an indole oxoacetic acid derivative (Elokdah et al., 2004). Using a cell based screen of 250 compounds, Sun et al. identified Tiplaxtinin as a bacterial cell division inhibitor (Sun et al., 2017c). Tiplaxtinin has strong antibacterial activity against Gram-positive pathogens, with MICs of 4.55–9.10 μM (2–4 μg/mL). Both in vitro and in vivo findings indicate that tiplaxtinin is capable of effectively disrupting dynamic assembly of FtsZ, GTPase function and Z-ring formation; tiplaxtinin-induced multiple FtsZ foci in B. subtilis cells is similar to the in vivo effects of benzamides. Molecular docking studies of this compound in SaFtsZ revealed that tiplaxtinin binds near the T7-loop and H7 helix in the IDC region. The trifluoromethoxy group of tiplaxtinin forms hydrogen bonds with G193 and G227 and halogen bonds with V189, Q192, G193, and M226 (Sun et al., 2017c). Similarly, the carbonyl group interacts with T265 through a hydrogen bond. Tiplaxtinin also interacts with V189, Q192, G193 D199, L200, L209, M226, G227, I228, and V297 residues via hydrophobic interactions (Table 4).