BackgroundGriseofulvin, an antifungal drug, has recently been shown to inhibit proliferation of various types of cancer cells and to inhibit tumor growth in athymic mice. Due to its low toxicity, griseofulvin has drawn considerable attention for its potential use in cancer chemotherapy. This work aims to understand how griseofulvin suppresses microtubule dynamics in living cells and sought to elucidate the antimitotic and antiproliferative action of the drug.MethodsThe effects of griseofulvin on the dynamics of individual microtubules in live MCF-7 cells were measured by confocal microscopy. Immunofluorescence microscopy, western blotting and flow cytometry were used to analyze the effects of griseofulvin on spindle microtubule organization, cell cycle progression and apoptosis. Further, interactions of purified tubulin with griseofulvin were studied in vitro by spectrophotometry and spectrofluorimetry. Docking analysis was performed using autodock4 and LigandFit module of Discovery Studio 2.1.ResultsGriseofulvin strongly suppressed the dynamic instability of individual microtubules in live MCF-7 cells by reducing the rate and extent of the growing and shortening phases. At or near half-maximal proliferation inhibitory concentration, griseofulvin dampened the dynamicity of microtubules in MCF-7 cells without significantly disrupting the microtubule network. Griseofulvin-induced mitotic arrest was associated with several mitotic abnormalities like misaligned chromosomes, multipolar spindles, misegregated chromosomes resulting in cells containing fragmented nuclei. These fragmented nuclei were found to contain increased concentration of p53. Using both computational and experimental approaches, we provided evidence suggesting that griseofulvin binds to tubulin in two different sites; one site overlaps with the paclitaxel binding site while the second site is located at the αβ intra-dimer interface. In combination studies, griseofulvin and vinblastine were found to exert synergistic effects against MCF-7 cell proliferation.ConclusionsThe study provided evidence suggesting that griseofulvin shares its binding site in tubulin with paclitaxel and kinetically suppresses microtubule dynamics in a similar manner. The results revealed the antimitotic mechanism of action of griseofulvin and provided evidence suggesting that griseofulvin alone and/or in combination with vinblastine may have promising role in breast cancer chemotherapy.

A perturbation of FtsZ assembly dynamics has been shown to inhibit bacterial cytokinesis. In this study, the antibacterial activity of 151 rhodanine compounds was assayed using Bacillus subtilis cells. Of 151 compounds, eight strongly inhibited bacterial proliferation at 2 μM. Subsequently, we used the elongation of B. subtilis cells as a secondary screen to identify potential FtsZ-targeted antibacterial agents. We found that three compounds significantly increased bacterial cell length. One of the three compounds, namely, CCR-11 [(E)-2-thioxo-5-({[3-(trifluoromethyl)phenyl]furan-2-yl}methylene)thiazolidin-4-one], inhibited the assembly and GTPase activity of FtsZ in vitro. CCR-11 bound to FtsZ with a dissociation constant of 1.5 ± 0.3 μM. A docking analysis indicated that CCR-11 may bind to FtsZ in a cavity adjacent to the T7 loop and that short halogen–oxygen, H-bonding, and hydrophobic interactions might be important for the binding of CCR-11 with FtsZ. CCR-11 inhibited the proliferation of B. subtilis cells with a half-maximal inhibitory concentration (IC50) of 1.2 ± 0.2 μM and a minimal inhibitory concentration of 3 μM. It also potently inhibited proliferation of Mycobacterium smegmatis cells. Further, CCR-11 perturbed Z-ring formation in B. subtilis cells; however, it neither visibly affected nucleoid segregation nor altered the membrane integrity of the cells. CCR-11 inhibited HeLa cell proliferation with an IC50 value of 18.1 ± 0.2 μM (∼15 × IC50 of B. subtilis cell proliferation). The results suggested that CCR-11 inhibits bacterial cytokinesis by inhibiting FtsZ assembly, and it can be used as a lead molecule to develop FtsZ-targeted antibacterial agents.

The assembly of FtsZ plays a central role in construction of the cytokinetic Z-ring that orchestrates bacterial cell division. A naturally occurring naphthoquinone, plumbagin, is known to exhibit antibacterial properties against several types of bacteria. In this study, plumbagin was found to perturb formation of the Z-ring in Bacillus subtilis 168 cells and to cause elongation of these cells without an apparent effect on nucleoid segregation, indicating that it may inhibit FtsZ assembly. Furthermore, it bound to purified B. subtilis FtsZ (BsFtsZ) with a dissociation constant of 20.7 AE 5.6 lM, and inhibited the assembly and GTPase activity of BsFtsZ in vitro. Interestingly, plumbagin did not inhibit either the assembly or GTPase activity of Escherichia coli FtsZ (EcFtsZ) in vitro. Using docking analysis, a putative plumbagin-binding site on BsFtsZ was identified, and the analysis indicated that hydrophobic interactions and hydrogen bonds predominate. Based on the in silico analysis, two variants of BsFtsZ, namely D199A and V307R, were constructed to explore the binding interaction of plumbagin and BsFtsZ. The effects of plumbagin on the assembly and GTPase activity of the variant BsFtsZ proteins in vitro indicated that the residues D199 and V307 may be involved in the binding of plumbagin to BsFtsZ. The results suggest that plumbagin inhibits bacterial proliferation by inhibiting the assembly of FtsZ, and provide insight into the binding site of plumbagin on BsFtsZ, which may help in the design of potent FtsZ-targeted antibacterial agents.

We have identified a potent antibacterial agent N‐(4‐sec‐butylphenyl)‐2‐(thiophen‐2‐yl)‐1H‐benzo[d]imidazole‐4‐carboxamide (BT‐benzo‐29) from a library of benzimidazole derivatives that stalled bacterial division by inhibiting FtsZ assembly. A short (5 min) exposure of BT‐benzo‐29 disassembled the cytokinetic Z‐ring in Bacillus subtilis cells without affecting the cell length and nucleoids. BT‐benzo‐29 also perturbed the localization of early and late division proteins such as FtsA, ZapA and SepF at the mid‐cell. Further, BT‐benzo‐29 bound to FtsZ with a dissociation constant of 24 ± 3 μm and inhibited the assembly and GTPase activity of purified FtsZ. A docking analysis suggested that BT‐benzo‐29 may bind to FtsZ at the C‐terminal domain near the T7 loop. BT‐benzo‐29 displayed significantly weaker inhibitory effects on the assembly and GTPase activity of two mutants (L272A and V275A) of FtsZ supporting the prediction of the docking analysis. Further, BT‐benzo‐29 did not appear to inhibit DNA duplication and nucleoid segregation and it did not perturb the membrane potential of B. subtilis cells. The results suggested that BT‐benzo‐29 exerts its potent antibacterial activity by inhibiting FtsZ assembly. Interestingly, BT‐benzo‐29 did not affect the membrane integrity of mammalian red blood cells. BT‐benzo‐29 bound to tubulin with a much weaker affinity than FtsZ and exerted significantly weaker effects on mammalian cells than on the bacterial cells indicating that the compound may have a strong antibacterial potential.

Recently, we found that divalent calcium has no detectable effect on the assembly of Mycobacterium tuberculosis FtsZ (MtbFtsZ), whereas it strongly promoted the assembly of Escherichia coli FtsZ (EcFtsZ). While looking for potential calcium binding residues in EcFtsZ, we found a mutation (E93R) that strongly promoted the assembly of EcFtsZ. The mutation increased the stability and bundling of the FtsZ protofilaments and produced a dominating effect on the assembly of the wild type FtsZ (WT-FtsZ). Although E93R-FtsZ was found to bind to GTP similarly to the WT-FtsZ, it displayed lower GTPase activity than the WT-FtsZ. E93R-FtsZ complemented for its wild type counterpart as observed by a complementation test using JKD7-1/pKD3 cells. However, the bacterial cells became elongated upon overexpression of the mutant allele. We modeled the structure of E93R-FtsZ using the structures of MtbFtsZ/Methanococcus jannaschi FtsZ (MjFtsZ) dimers as templates. The MtbFtsZ-based structure suggests that the Arg 93 -Glu 138 salt bridge provides the additional stability, whereas the effect of mutation appears to be indirect (allosteric) if the EcFtsZ dimer is similar to that of MjFtsZ. The data presented in this study suggest that an increase in the stability of the FtsZ protofilaments is detrimental for the bacterial cytokinesis.Cell division in almost all bacteria is primarily driven by FtsZ, a protein considered as the homologue of the eukaryotic cytoskeletal protein tubulin (1, 2). FtsZ is a highly conserved protein in prokaryotes and plays a key role in the spatiotemporal regulation of the divisome assembly (2). Polymerization of FtsZ results in the formation of a ring-like structure, known as the Z-ring. The Z-ring is a highly dynamic structure (3), and the dynamics of the Z-ring is attributed to the assembly and disassembly of FtsZ protofilaments (4). Perturbation of the FtsZ assembly has been reported to impair bacterial cell division (5-9).Spatiotemporal regulation of the Z-ring at the midcell is tightly regulated by several effectors of FtsZ assembly (10 -12). Although the arrangement of FtsZ polymers in the bacterial Z-ring has not yet been visualized, the reconstitution of the FtsZ polymers in vitro has contributed a lot to our understanding of the bacterial cell division. Recently, an attempt was made to reconstitute the Z-ring in a liposome in vitro, and it was found that neither FtsZ assembly nor the generation of the constriction force requires FtsA or other downstream division proteins (13). Further, FtsZ polymers have been found to have intrinsic ability to form cytoplasmic ringlike structures in yeast cells, indicating that FtsZ assembly into the ring may not require any other regulatory protein (14).FtsZ protein of different bacteria exhibit different assembly properties in vitro. For example, the polymerization of Escherichia coli FtsZ (EcFtsZ) 2 has been observed to be more rapid than that of the Mycobacterium tuberculosis FtsZ (MtbFtsZ) (15). Recently, we have shown that divalent calcium strongly influenced ...

The article suggests that vaccination with tubulin may offer novel opportunities for the antiparasitic treatment. Native or recombinant tubulin used as antigen has been shown to elicit immune response and cure infection partially or fully in animals upon challenge by protozoan parasites and helminths, thus indicating the suitability of tubulin as a vaccine against parasitic diseases.

Insights into the functioning of the centrosomal proteins will be extremely beneficial in validating the centrosome as a target in cancer therapy. New strategies either as a single entity or in combination with current chemotherapeutic agents should be researched or exploited to reveal the promises that the centrosome holds for future cancer therapy.

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