Fluorescence spectroscopic methods to analyze drug-tubulin interactions.
TL;DR: This chapter has discussed its use to characterize the binding parameters of ligands that are known to bind to three important sites in tubulin namely the vinca domain, the colchicine binding site, and the taxol site.
Abstract: Fluorescence spectroscopy has been extensively used to characterize ligand binding to tubulin and microtubules The inherent advantages of fluorescence spectroscopic methods lie in their ease, sensitivity to local environmental changes, and ability to describe the protein-ligand interactions qualitatively as well as quantitatively in equilibrium conditions In this chapter, we have described how fluorescence spectroscopy has been used to decipher molecular interaction between a wide variety of ligands and tubulin Particularly, we have discussed its use to characterize the binding parameters of ligands that are known to bind to three important sites in tubulin namely the vinca domain, the colchicine binding site, and the taxol site These are the sites where most of the microtubule-targeted anticancer agents bind to tubulin An understanding of the interaction between tubulin and small molecule inhibitors can assist in understanding the cellular effects of these inhibitors This will also help in developing molecules that have higher binding affinity to tubulin and can serve as potent anticancer agents
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TL;DR: Plumbagin inhibits bacterial proliferation by inhibiting the assembly of FtsZ, and insight into the binding site of plumbagin on BsFtsZ is provided, which may help in the design of potent FTSZ‐targeted antibacterial agents.
Abstract: 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.
49 citations
TL;DR: These findings explore the cytotoxic potential of NMK-BH3 by targeting the microtubules and inspire its development as a potential candidate for lung cancer chemotherapy.
Abstract: The biological significance of microtubules makes them a validated target of cancer therapy. In this study, we have utilized indole, an important pharmacological scaffold, to synthesize novel bis(indolyl)-hydrazide–hydrazone derivatives (NMK-BH compounds) and recognized NMK-BH3 as the most effective one in inhibiting A549 cell proliferation and assembly of tissue-purified tubulin. Cell viability experiments showed that NMK-BH3 inhibited proliferation of human lung adenocarcinoma (A549) cells, normal human lung fibroblasts (WI38) and peripheral blood mononuclear cells (PBMC) with IC50 values of ∼2, 48.5, and 62 μM, respectively. Thus, the relatively high cytotoxicity of NMK-BH3 toward lung carcinoma (A549) cells over normal lung fibroblasts (WI38) and PBMC confers a therapeutic advantage of reduced host toxicity. Flow cytometry, Western blot, and immunofluorescence studies in the A549 cell line revealed that NMK-BH3 induced G2/M arrest, mitochondrial depolarization, and apoptosis by depolymerizing the cell...
45 citations
TL;DR: CXI-benzo-84 inhibited cell cycle progression in metaphase stage of mitosis and accumulated spindle assembly checkpoint proteins Mad2 and BubR1 on kinetochores, which subsequently activated apoptotic cell death in cancer cells.
43 citations
TL;DR: This report describes how AOPs could be harnessed to advance MOA analysis of genotoxicity pathways using five example case studies and illustrates the practical considerations and evidence required to formalize such Aops so that they may be applied to genetic toxicity evaluation schemes.
Abstract: In May 2017, the Health and Environmental Sciences Institute's Genetic Toxicology Technical Committee hosted a workshop to discuss whether mode of action (MOA) investigation is enhanced through the application of the adverse outcome pathway (AOP) framework. As AOPs are a relatively new approach in genetic toxicology, this report describes how AOPs could be harnessed to advance MOA analysis of genotoxicity pathways using five example case studies. Each of these genetic toxicology AOPs proposed for further development includes the relevant molecular initiating events, key events, and adverse outcomes (AOs), identification and/or further development of the appropriate assays to link an agent to these events, and discussion regarding the biological plausibility of the proposed AOP. A key difference between these proposed genetic toxicology AOPs versus traditional AOPs is that the AO is a genetic toxicology endpoint of potential significance in risk characterization, in contrast to an adverse state of an organism or a population. The first two detailed case studies describe provisional AOPs for aurora kinase inhibition and tubulin binding, leading to the common AO of aneuploidy. The remaining three case studies highlight provisional AOPs that lead to chromosome breakage or mutation via indirect DNA interaction (inhibition of topoisomerase II, production of cellular reactive oxygen species, and inhibition of DNA synthesis). These case studies serve as starting points for genotoxicity AOPs that could ultimately be published and utilized by the broader toxicology community and illustrate the practical considerations and evidence required to formalize such AOPs so that they may be applied to genetic toxicity evaluation schemes. Environ. Mol. Mutagen. 61:114-134, 2020. © 2019 Wiley Periodicals, Inc.
41 citations
Cites background from "Fluorescence spectroscopic methods ..."
...The binding mechanism of a number of tubulin binders has been studied in great detail and can be easily studied by fluorescence spectroscopic methods (for reference, see Bhattacharyya et al., 2010)....
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TL;DR: The results suggested that BCFMT inhibits proliferation of several types of cancer cells including drug resistance cells by suppressing microtubule dynamics and indicated that the compound may have chemotherapeutic potential.
Abstract: Using cell based screening assay, we identified a novel anti-tubulin agent (Z)-5-((5-(4-bromo-3-chlorophenyl)furan-2-yl)methylene)-2-thioxothiazoli din-4-one (BCFMT) that inhibited proliferation of human cervical carcinoma (HeLa) (IC50, 7.2 +/- 1.8 mu M), human breast adenocarcinoma (MCF-7) (IC50, 10.0 +/- 0.5 mu M), highly metastatic breast adenocarcinoma (MDA-MB-231) (IC50, 6.0 +/- 1 mu M), cisplatin-resistant human ovarian carcinoma (A2780-cis) (IC50, 5.8 +/- 0.3 mu M) and multi-drug resistant mouse mammary tumor (EMT6/AR1) (IC50, 6.5 +/- 1 mu M) cells. Using several complimentary strategies, BCFMT was found to inhibit cancer cell proliferation at G2/M phase of the cell cycle apparently by targeting microtubules. In addition, BCFMT strongly suppressed the dynamics of individual microtubules in live MCF-7 cells. At its half maximal proliferation inhibitory concentration (10 mu M), BCFMT reduced the rates of growing and shortening phases of microtubules in MCF-7 cells by 37 and 40%, respectively. Further, it increased the time microtubules spent in the pause (neither growing nor shortening detectably) state by 135% and reduced the dynamicity (dimer exchange per unit time) of microtubules by 70%. In vitro, BCFMT bound to tubulin with a dissociation constant of 8.3 +/- 1.8 mu M, inhibited tubulin assembly and suppressed GTPase activity of microtubules. BCFMT competitively inhibited the binding of BODIPY FL-vinblastine to tubulin with an inhibitory concentration (K-i) of 5.2 +/- 1.5 mu M suggesting that it binds to tubulin at the vinblastine site. In cultured cells, BCFMT-treatment depolymerized interphase microtubules, perturbed the spindle organization and accumulated checkpoint proteins (BubR1 and Mad2) at the kinetochores. BCFMT-treated MCF-7 cells showed enhanced nuclear accumulation of p53 and its downstream p21, which consequently activated apoptosis in these cells. The results suggested that BCFMT inhibits proliferation of several types of cancer cells including drug resistance cells by suppressing microtubule dynamics and indicated that the compound may have chemotherapeutic potential.
34 citations
Cites methods from "Fluorescence spectroscopic methods ..."
...The intrinsic tryptophan fluorescence of tubulin has been widely used to determine the binding constant of a ligand to tubulin [37]....
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References
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Book•
01 Jan 1983
TL;DR: This book describes the fundamental aspects of fluorescence, the biochemical applications of this methodology, and the instrumentation used in fluorescence spectroscopy.
Abstract: Fluorescence methods are being used increasingly in biochemical, medical, and chemical research. This is because of the inherent sensitivity of this technique. and the favorable time scale of the phenomenon of fluorescence. 8 Fluorescence emission occurs about 10- sec (10 nsec) after light absorp tion. During this period of time a wide range of molecular processes can occur, and these can effect the spectral characteristics of the fluorescent compound. This combination of sensitivity and a favorable time scale allows fluorescence methods to be generally useful for studies of proteins and membranes and their interactions with other macromolecules. This book describes the fundamental aspects of fluorescence. and the biochemical applications of this methodology. Each chapter starts with the -theoreticalbasis of each phenomenon of fluorescence, followed by examples which illustrate the use of the phenomenon in the study of biochemical problems. The book contains numerous figures. It is felt that such graphical presentations contribute to pleasurable reading and increased understand ing. Separate chapters are devoted to fluorescence polarization, lifetimes, quenching, energy transfer, solvent effects, and excited state reactions. To enhance the usefulness of this work as a textbook, problems are included which illustrate the concepts described in each chapter. Furthermore, a separate chapter is devoted to the instrumentation used in fluorescence spectroscopy. This chapter will be especially valuable for those perform ing or contemplating fluorescence measurements. Such measurements are easily compromised by failure to consider a number of simple principles."
28,073 citations
"Fluorescence spectroscopic methods ..." refers background or methods in this paper
...Here, K(2) was assumed to be 2/3 (Lakowicz, 1999) and the refractive index of water (1....
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...The emission intensity of DA at 390 nm was corrected for inner filter effect as described by Lakowicz (1999) using the equation Fcorr ¼ Fobs antilog ODexþODem 2 where Fcorr is inner filter corrected emission intensity of DA, Fobs is the observed emission intensity, ODex is the absorption of the DA…...
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...Their quantum yields increase several folds upon binding to proteins (Lakowicz, 1999)....
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...Samples with fixed concentration of tubulin and increasing concentration of ligand are prepared separately; the fluorescence is usually measured using 2 10mm quartz cuvette and the inner filter correction is made using the equation of Lakowicz (1999)....
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...Here, K2 was assumed to be 2/3 (Lakowicz, 1999) and the refractive index of water (1.33) was used for calculation....
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TL;DR: Highly dynamic mitotic-spindle microtubules are among the most successful targets for anticancer therapy, and it is now known that at lower concentrations, microtubule-targeted drugs can suppress micro Tubule dynamics without changingmicrotubule mass; this action leads to mitotic block and apoptosis.
Abstract: Highly dynamic mitotic-spindle microtubules are among the most successful targets for anticancer therapy. Microtubule-targeted drugs, including paclitaxel and Vinca alkaloids, were previously considered to work primarily by increasing or decreasing the cellular microtubule mass. Although these effects might have a role in their chemotherapeutic actions, we now know that at lower concentrations, microtubule-targeted drugs can suppress microtubule dynamics without changing microtubule mass; this action leads to mitotic block and apoptosis. In addition to the expanding array of chemically diverse antimitotic agents, some microtubule-targeted drugs can act as vascular-targeting agents, rapidly depolymerizing microtubules of newly formed vasculature to shut down the blood supply to tumours.
4,007 citations
"Fluorescence spectroscopic methods ..." refers background in this paper
...They alter microtubule dynamics at nanomolar concentrations (Panda et al., 1996; Jordan and Wilson, 2004), inhibit microtubule assembly at micromolar concentrations, induce aggregation and paracrystal formation at high concentrations (Himes, 1991; Warfield and Bouck, 1974; Wilson et al., 1982), and…...
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...They alter microtubule dynamics at nanomolar concentrations (Panda et al., 1996; Jordan and Wilson, 2004), inhibit microtubule assembly...
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TL;DR: Changes in the subunits of tubulin as it switches from its straight conformation to a curved one correlate with the loss of lateral contacts and provide a rationale for the rapid microtubule depolymerization characteristic of dynamic instability.
Abstract: Microtubules are cytoskeletal polymers of tubulin involved in many cellular functions. Their dynamic instability is controlled by numerous compounds and proteins, including colchicine and stathmin family proteins. The way in which microtubule instability is regulated at the molecular level has remained elusive, mainly because of the lack of appropriate structural data. Here, we present the structure, at 3.5 A resolution, of tubulin in complex with colchicine and with the stathmin-like domain (SLD) of RB3. It shows the interaction of RB3-SLD with two tubulin heterodimers in a curved complex capped by the SLD amino-terminal domain, which prevents the incorporation of the complexed tubulin into microtubules. A comparison with the structure of tubulin in protofilaments shows changes in the subunits of tubulin as it switches from its straight conformation to a curved one. These changes correlate with the loss of lateral contacts and provide a rationale for the rapid microtubule depolymerization characteristic of dynamic instability. Moreover, the tubulin-colchicine complex sheds light on the mechanism of colchicine's activity: we show that colchicine binds at a location where it prevents curved tubulin from adopting a straight structure, which inhibits assembly.
1,418 citations
"Fluorescence spectroscopic methods ..." refers background in this paper
...Binding sites for these molecules on tubulin are well characterized and the crystal structures of their complexes with tubulin have been reported (Gigant et al., 2005; Nogales et al., 1995; Ravelli et al., 2004)....
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Journal Article•
TL;DR: Epothilones represent a novel structural class of compounds, the first to be described since the original discovery ofTaxol, which not only mimic the biological effects of taxol but also appear to bind to the same microtubule-binding site as taxol.
Abstract: Tubulin polymerization into microtubules is a dynamic process, with the equilibrium between growth and shrinkage being essential for many cellular processes. The antineoplastic agent taxol hyperstabilizes polymerized microtubules, leading to mitotic arrest and cytotoxicity in proliferating cells. Using a sensitive filtration-calorimetric assay to detect microtubule nucleating activity, we have identified epothilones A and B as compounds that possess all the biological effects of taxol both in vitro and in cultured cells. The epothilones are equipotent and exhibit kinetics similar to taxol in inducing tubulin polymerization into microtubules in vitro (filtration, light scattering, sedimentation, and electron microscopy) and in producing enhanced microtubule stability and bundling in cultured cells. Furthermore, these 16-membered macrolides are competitive inhibitors of [3H]taxol binding, exhibiting a 50% inhibitory concentration almost identical to that of taxol in displacement competition assays. Epothilones also cause cell cycle arrest at the G2-M transition leading to cytotoxicity, similar to taxol. In contrast to taxol, epothilones retain a much greater toxicity against P-glycoprotein-expressing multiple drug resistant cells. Epothilones, therefore, represent a novel structural class of compounds, the first to be described since the original discovery of taxol, which not only mimic the biological effects of taxol but also appear to bind to the same microtubule-binding site as taxol.
1,188 citations
"Fluorescence spectroscopic methods ..." refers background in this paper
...E. Analysis of the Environment of the Binding Site of Taxol on Microtubules The taxoid binding site in tubulin has been suggested to be the binding site for a large number of molecules including epothilones and discodermolide (Bollag et al., 1995; Buey et al., 2005)....
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TL;DR: A high-resolution model of the microtubule has been obtained by docking the crystal structure of tubulin into a 20 A map of themicrotubule, and the excellent fit indicates the similarity of the tubulin conformation in both polymers and defines the orientation of the Tubulin structure within the micro Tubule.
1,135 citations
"Fluorescence spectroscopic methods ..." refers background in this paper
...Although the crystal structure of taxol-stabilized tubulin sheets has indicated that the taxol-binding site faces the inner lumen of microtubules (Nogales et al., 1999), the kinetic studies with the fluorescent taxol analogues have suggested otherwise (Diaz et al., 2003, 2005)....
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