BT-benzo-29 inhibits bacterial cell proliferation by perturbing FtsZ assembly.

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.

The centrosome: a prospective entrant in cancer therapy.

INTRODUCTION: The centrosome plays an essential role in the cell cycle. The centrosome and its associated proteins assist in nucleating and organizing microtubules. A structural or a functional aberration in the centrosome is known to cause abnormal cell proliferation leading to tumors. Therefore, the centrosome is considered as a promising anti-cancer target. AREAS COVERED: This review begins with a brief introduction to the centrosome and its role in the cell cycle. We elaborate on the centrosome-associated proteins that regulate microtubule dynamics. In addition, we discuss the centrosomal protein kinase targets such as cyclin-dependent, polo-like and aurora kinases. Inhibitors targeting these kinases are undergoing clinical trials for cancer chemotherapy. Further, we shed light on new approaches to target the centrosomal proteins for cancer therapy. EXPERT OPINION: 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.

Understanding FtsZ assembly: cues from the behavior of its N- and C-terminal domains.

FtsZ polymerizes to form a cytokinetic ring at the center of a bacterial cell, which engineers bacterial cell division. FtsZ consists of N-terminal and C-terminal core domains followed by a C-terminal spacer and a conserved C-terminal tail region. Though it has been reported that both N- and C-domains can fold independently, the assembly behaviors of the N- and C-domains are not clear. In this study, we created five truncated constructs of Bacillus subtilis FtsZ, two N-domain and three C-domain constructs, and expressed and purified them. We determined their assembly properties and their effect on the assembly of full-length FtsZ to gain insight into the mechanism of FtsZ polymerization. We found that the N-domain of B. subtilis FtsZ can polymerize on its own in a GTP-dependent manner. Further, we obtained evidence indicating that the N-domain could bind to GTP but could not hydrolyze GTP by itself. In addition, the N-domain was found to inhibit the assembly of full-length FtsZ. Interestingly, the N-domain was found to enhance the GTPase activity of full-length FtsZ. An analysis of the effects of the N- and C-domains on FtsZ assembly indicated that the assembly of FtsZ might be directional. The work has provided new insight into the assembly characteristics of FtsZ domains and the mechanism of FtsZ polymerization.

Plumbagin inhibits cytokinesis in Bacillus subtilis by inhibiting FtsZ assembly--a mechanistic study of its antibacterial activity.

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 ± 5.6 µM, 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.

A rhodanine derivative CCR-11 inhibits bacterial proliferation by inhibiting the assembly and GTPase activity of FtsZ.

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 (IC(50)) 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 IC(50) value of 18.1 ± 0.2 µM (∼15 × IC(50) 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.

Microtubules as antifungal and antiparasitic drug targets.

INTRODUCTION: Diseases caused by fungi and parasites are major illnesses in humans as well as in animals. Microtubule-targeted drugs are highly effective for the treatment of fungal and parasitic infections; however, several human parasitic infections such as malaria, trypanosomiasis and leishmaniasis do not have effective remedial drugs. In addition, the emergence of drug-resistant fungi and parasites makes the discovery of new drugs imperative. AREAS COVERED: This article describes similarities and dissimilarities between parasitic, fungal and mammalian tubulins and focuses on microtubule-targeting agents and therapeutic approaches for the treatment of fungal and parasitic diseases. New microtubule-targeted antileishmanial, antimalarial and antifungal drugs, with structures, biological activities and related patents, are described. The potential of dsRNA against tubulin to inhibit proliferation of protozoan and helminthic parasites is also discussed. Patent documents up to 2010 have been searched on USPTO, Patentscope, and Espacenet resources. EXPERT OPINION: 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.

E93R substitution of Escherichia coli FtsZ induces bundling of protofilaments, reduces GTPase activity, and impairs bacterial cytokinesis.

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.

Griseofulvin stabilizes microtubule dynamics, activates p53 and inhibits the proliferation of MCF-7 cells synergistically with vinblastine.

BACKGROUND: Griseofulvin, 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. METHODS: The 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. RESULTS: Griseofulvin 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 alphabeta intra-dimer interface. In combination studies, griseofulvin and vinblastine were found to exert synergistic effects against MCF-7 cell proliferation. CONCLUSIONS: The 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.