Dulal Panda

Publications of Dulal Panda

• Pyrophosphate sensing by a fluorescent Zn2+ bound triazole linked imino-thiophenyl conjugate of calix[4]arene in HEPES buffer medium: Spectroscopy, microscopy and cellular studies.

  Authors: Chebrolu Pulla Rao, Rakesh Kumar Pathak, Khatija Tabbasum, Ankit Rai, Dulal Panda

  Analytical chemistry. 05/2012;

  An in situ prepared Zn2+ complex of triazole linked imino-thiophenyl conjugate of calix[4]arene, [ZnL] was demonstrated to be highly fluorescent in HEPES buffer solution. [ZnL] has been used asAn in situ prepared Zn2+ complex of triazole linked imino-thiophenyl conjugate of calix[4]arene, [ZnL] was demonstrated to be highly fluorescent in HEPES buffer solution. [ZnL] has been used as chemosensing ensemble for the recognition of phosphates in general and pyrophosphates in particular among the eighteen different anions studied. The chemosensing behavior of the [ZnL] has been demonstrated through fluorescence, absorption, visual fluorescent color changes, ESI MS and 1H NMR titrations. Variations in the micro structural features of L, its zinc complex and the complex upon addition of PPi have been demonstrated through atomic force microscopy and transmission electron microscopy. Such studies have been extended to see the permeability of the conjugate in to the Hela cells by fluorescence microscopy. In accession, a reversible "write-read-erase-read" logic gate property of L has been demonstrated through a feedback loop in the presence of Zn2+ and PPi.

• Imino-Phenolic-Pyridyl Conjugates of Calix[4]arene (L(1) and L(2)) as Primary Fluorescence Switch-on Sensors for Zn(2+) in Solution and in HeLa Cells and the Recognition of Pyrophosphate and ATP by [ZnL(2)].

  Authors: Rakesh Kumar Pathak, Vijaya Kumar Hinge, Ankit Rai, Dulal Panda, Chebrolu Pulla Rao

  Inorganic chemistry. 04/2012;

  Pyridyl-based triazole-linked calix[4]arene conjugates, viz. L(1) and L(2), were synthesized and characterized. These two conjugates were shown to be selective and sensitive for Zn(2+) among the 12Pyridyl-based triazole-linked calix[4]arene conjugates, viz. L(1) and L(2), were synthesized and characterized. These two conjugates were shown to be selective and sensitive for Zn(2+) among the 12 metal ions studied in HEPES buffer medium by fluorescence, absorption, and visual color change with the detection limit of ∼31 and ∼112 ppb, respectively, by L(1) and L(2). Moreover, the utility of the conjugates L(1) and L(2) in showing the zinc recognition in live cells has also been demonstrated using HeLa cells as monitored by fluorescence imaging. The zinc complexes of L(1) and L(2) were isolated, and the structure of [ZnL(1)] has been established by single-crystal XRD and that of [ZnL(2)] by DFT calculations. TDDFT calculations were performed in order to demonstrate the electronic properties of receptors and their zinc complexes. The isolated zinc complexes, viz. [ZnL(1)] and [ZnL(2)], have been used as molecular tools for the recognition of anions on the basis of their binding affinities toward Zn(2+). [ZnL(2)] was found to be sensitive and selective toward phosphate-bearing ions and molecules and in particular to pyrophosphate (PPi) and ATP among the other 18 anions studied; however, [ZnL(1)] was not sensitive toward any of the anions studied. The selectivity has been shown on the basis of the changes observed in the emission and absorption spectral studies through the removal of Zn(2+) from [ZnL(2)] by PPi. Thus, [ZnL(2)] has been shown to detect PPi up to 278 ± 10 ppb at pH 7.4 in aqueous methanolic (1/2 v/v) HEPES buffer.

• Kinetic stabilization of microtubule dynamics by indanocine perturbs EB1 localization, induces defects in cell polarity and inhibits migration of MDA-MB-231 cells.

  Authors: Sonia Kapoor, Dulal Panda

  Biochemical pharmacology. 02/2012; 83(11):1495-506.

  Cell motility is an essential aspect of metastatic spread of cancer. Microtubule-targeted agents exhibit anti-metastatic properties, the underlying mechanism of which remains understudied. In thisCell motility is an essential aspect of metastatic spread of cancer. Microtubule-targeted agents exhibit anti-metastatic properties, the underlying mechanism of which remains understudied. In this study, we have investigated the role of microtubule dynamics in migration of cancer cells using indanocine, a synthetic small molecule inhibitor of tubulin. We found that indanocine, at concentrations that did not visibly affect microtubule organization, suppressed dynamic instability of microtubules and reduced the rate of migration of highly metastatic MDA-MB-231 cells. Indanocine-treated cells were defective in lamellipodium formation and could not develop polarized morphology. The kinetic stabilization of microtubules was associated with a marked increase in their acetylation level and a perturbation in the localization of EB1, a microtubule plus end binding protein. Using standard scratch wound healing assay and immunofluorescence analysis; we found that microtubule acetylation occurred in the direction of migration in vehicle-treated cells, whereas indanocine treatment led to a global acetylation of microtubules. The results together suggested that selective stabilization of microtubules was perturbed in the presence of indanocine that possibly resulted in lack of cell polarization and a concurrent reduction in migration of cells. Moreover, microtubule stabilization by indanocine affected adhesion turnover and impaired the polarized pattern of adhesion sites in cells. Together the results indicated that the regulation of microtubule dynamics is required to coordinate cell polarization as well as adhesion asymmetry and support the hypothesis that the perturbation of microtubule dynamics by tubulin-targeted agents can be exploited to restrict the migration of tumor cells.

• Curcumin recognizes a unique binding site of tubulin.

  Authors: Soumyananda Chakraborti, Lalita Das, Neha Kapoor, Amlan Das, Vishnu Dwivedi, Asim Poddar, Gopal Chakraborti, Mark Janik, Gautam Basu, Dulal Panda, Pinak Chakrabarti, Avadhesha Surolia, Bhabatarak Bhattacharyya

  Journal of medicinal chemistry. 08/2011; 54(18):6183-96.

  Although curcumin is known for its anticarcinogenic properties, the exact mechanism of its action or the identity of the target receptor is not completely understood. Studies on a series of curcuminAlthough curcumin is known for its anticarcinogenic properties, the exact mechanism of its action or the identity of the target receptor is not completely understood. Studies on a series of curcumin analogues, synthesized to investigate their tubulin binding affinities and tubulin self-assembly inhibition, showed that: (i) curcumin acts as a bifunctional ligand, (ii) analogues with substitution at the diketone and acetylation of the terminal phenolic groups of curcumin are less effective, (iii) a benzylidiene derivative, compound 7, is more effective than curcumin in inhibiting tubulin self-assembly. Cell-based studies also showed compound 7 to be more effective than curcumin. Using fluorescence spectroscopy we show that curcumin binds tubulin 32 Å away from the colchicine-binding site. Docking studies also suggests that the curcumin-binding site to be close to the vinblastine-binding site. Structure-activity studies suggest that the tridented nature of compound 7 is responsible for its higher affinity for tubulin compared to curcumin.

• Cationic lipid enhances assembly of bacterial cell division protein FtsZ: a possible role of bacterial membrane in FtsZ assembly dynamics.

  Authors: Anuradha Kuchibhatla, Jayesh Bellare, Dulal Panda

  International journal of biological macromolecules. 07/2011; 49(4):737-41.

  The assembly of FtsZ plays an important role in bacterial cell division. Lipids in the bacterial cell membrane have been suggested to play a role in directing the site of FtsZ assembly. Using lipidThe assembly of FtsZ plays an important role in bacterial cell division. Lipids in the bacterial cell membrane have been suggested to play a role in directing the site of FtsZ assembly. Using lipid monolayer and bilayer (liposome) systems, we directly examined the effects of cationic lipids on FtsZ assembly. We found that cationic lipids enhanced the assembly of FtsZ in association with an increase in the GTPase activity of FtsZ. The system consisting of lipid monolayer and bilayer (liposome) may mimic the bacterial membrane and therefore, the data might indicate the influence of bacterial membrane on the assembly of FtsZ protofilaments.

• Microtubules as antifungal and antiparasitic drug targets.

  Authors: Biswa Prasun Chatterji, Bhavya Jindal, Sanjeeva Srivastava, Dulal Panda

  Expert opinion on therapeutic patents. 02/2011; 21(2):167-86.

  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 parasiticINTRODUCTION: 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.

• ZipA binds to FtsZ with high affinity and enhances the stability of FtsZ protofilaments.

  Authors: Anuradha Kuchibhatla, Anusri Bhattacharya, Dulal Panda

  PloS one. 01/2011; 6(12):e28262.

  A bacterial membrane protein ZipA that tethers FtsZ to the membrane is known to promote FtsZ assembly. In this study, the binding of ZipA to FtsZ was monitored using fluorescence spectroscopy. ZipAA bacterial membrane protein ZipA that tethers FtsZ to the membrane is known to promote FtsZ assembly. In this study, the binding of ZipA to FtsZ was monitored using fluorescence spectroscopy. ZipA was found to bind to FtsZ with high affinities at three different (6.0, 6.8 and 8.0) pHs, albeit the binding affinity decreased with increasing pH. Further, thick bundles of FtsZ protofilaments were observed in the presence of ZipA under the pH conditions used in this study indicating that ZipA can promote FtsZ assembly and stabilize FtsZ polymers under unfavorable conditions. Bis-ANS, a hydrophobic probe, decreased the interaction of FtsZ and ZipA indicating that the interaction between FtsZ and ZipA is hydrophobic in nature. ZipA prevented the dilution induced disassembly of FtsZ polymers suggesting that it stabilizes FtsZ protofilaments. Fluorescein isothiocyanate-labeled ZipA was found to be uniformly distributed along the length of the FtsZ protofilaments indicating that ZipA stabilizes FtsZ protofilaments by cross-linking them.

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

  Authors: Richa Jaiswal, Ronak Y Patel, Jayant Asthana, Bhavya Jindal, Petety V Balaji, Dulal Panda

  The Journal of biological chemistry. 10/2010; 285(41):31796-805.

  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 FtsZRecently, 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.

• Dinuclear copper(I) complexes containing cyclodiphosphazane derivatives and pyridyl ligands: synthesis, structural studies, and antiproliferative activity toward human cervical and breast cancer cells.

  Authors: Maravanji S Balakrishna, D Suresh, Ankit Rai, Joel T Mague, Dulal Panda

  Inorganic chemistry. 10/2010; 49(19):8790-801.

  Several mixed-ligand copper(I) complexes of cyclodiphosphazanes, [(t)BuNP(NC(4)H(8)X)](2) (1, X = O; 2, X = NMe), were synthesized by reacting the octanuclear copper(I) complexesSeveral mixed-ligand copper(I) complexes of cyclodiphosphazanes, [(t)BuNP(NC(4)H(8)X)](2) (1, X = O; 2, X = NMe), were synthesized by reacting the octanuclear copper(I) complexes [Cu(8)(μ(2)-I)(8){[(t)BuNP(NC(4)H(8)X)](2)}(4)] (3, X = O; 4, X = NMe) with various pyridyl ligands. Interaction of the metallomacrocyclic complex 3 or 4 with pyridine, 2,2'-bipyridine, and 1,10-phenanthroline afforded the neutral dinuclear complexes [(C(5)H(5)N)(4)Cu(2)I(2){[(t)BuNP(NC(4)H(8)X)](2)}] (5, X = O; 6, X = NMe), [(2,2'-bpy)(2)Cu(2)I(2){[(t)BuNP(NC(4)H(8)X)](2)}] (7, X = O; 8, X = NMe), and [(1,10-phen)(2)Cu(2)I(2){[(t)BuNP(NC(4)H(8)X)](2)}] (9, X = O; 10, X = NMe), respectively, in good yield. The new dinuclear complexes 3, 5, and 7-9 were tested for their cytotoxic properties against human cervical cancer (HeLa) cells. The results indicated that all of the copper complexes have in vitro antitumor activity either similar to or better than that of cisplatin, a widely used anticancer drug. Among the compounds tested, complex 9 showed the most potent inhibitory activity in HeLa cells. In addition, complex 9 was found to potently inhibit proliferation of human breast cancer cells (MCF-7), highly metastatic breast cancer cells (MDA-MB 231), and nontransformed Chinese hamster ovary (CHO) cells. Complex 9 inhibited proliferation of these cells in culture more potently than cisplatin; for example, complex 9 was found to inhibit proliferation of HeLa and MCF-7 cells 3 and 5 times more efficiently than cisplatin. Complex 9 treatment damaged the DNA integrity, blocked the cells in the G1 phase of the cell cycle, and induced apoptosis via a p53-dependent pathway. The molecular structures of complexes 9 and 10 were confirmed by single-crystal X-ray diffraction studies.

• Corrigendum to "HMBA depolymerizes microtubules, activates mitotic checkpoints and induces mitotic block in MCF-7 cells by binding at the colchicine site in tubulin" [Biochem. Pharmacol. 80 (2010) 50-61].

  Authors: Biswa Prasun Chatterji, Mithu Banerjee, Parminder Singh, Dulal Panda

  Biochemical pharmacology. 10/2010; 80(7):1113.

• Probing the binding site of curcumin in Escherichia coli and Bacillus subtilis FtsZ--a structural insight to unveil antibacterial activity of curcumin.

  Authors: Simranjeet Kaur, Niraj H Modi, Dulal Panda, Nilanjan Roy

  European journal of medicinal chemistry. 09/2010; 45(9):4209-14.

  The cytoskeletal protein, FtsZ plays a pivotal role in prokaryotic cell division and is present in majority of the bacterial species. In recent years, inhibitors of FtsZ have been identified that mayThe cytoskeletal protein, FtsZ plays a pivotal role in prokaryotic cell division and is present in majority of the bacterial species. In recent years, inhibitors of FtsZ have been identified that may function as lead compounds for the development of novel antimicrobials. It has been found that curcumin, the main bioactive component of Curcuma longa, inhibits Bacillus subtilis and Escherichia coli growth by inhibiting FtsZ assembly. Though it is experimentally established that curcumin inhibits FtsZ polymerization, the binding site of curcumin in FtsZ is not known. In this study, interaction of curcumin with catalytic core domain of E. coli and B. subtilis FtsZ was investigated using computational docking.

• Curcumin suppresses the dynamic instability of microtubules, activates the mitotic checkpoint and induces apoptosis in MCF-7 cells.

  Authors: Mithu Banerjee, Parminder Singh, Dulal Panda

  The FEBS journal. 08/2010; 277(16):3437-48.

  In this study, curcumin, a potential anticancer agent, was found to dampen the dynamic instability of individual microtubules in living MCF-7 cells. It strongly reduced the rate and extent ofIn this study, curcumin, a potential anticancer agent, was found to dampen the dynamic instability of individual microtubules in living MCF-7 cells. It strongly reduced the rate and extent of shortening states, and modestly reduced the rate and extent of growing states. In addition, curcumin decreased the fraction of time microtubules spent in the growing state and strongly increased the time microtubules spent in the pause state. Brief treatment with curcumin depolymerized mitotic microtubules, perturbed microtubule-kinetochore attachment and disturbed the mitotic spindle structure. Curcumin also perturbed the localization of the kinesin protein Eg5 and induced monopolar spindle formation. Further, curcumin increased the accumulation of Mad2 and BubR1 at the kinetochores, indicating that it activated the mitotic checkpoint. In addition, curcumin treatment increased the metaphase/anaphase ratio, indicating that it can delay mitotic progression from the metaphase to anaphase. We provide evidence suggesting that the affected cells underwent apoptosis via the p53-dependent apoptotic pathway. The results support the idea that kinetic stabilization of microtubule dynamics assists in the nuclear translocation of p53. Curcumin exerted additive effects when combined with vinblastine, a microtubule depolymerizing drug, whereas the combination of curcumin with paclitaxel, a microtubule-stabilizing drug, produced an antagonistic effect on the inhibition of MCF-7 cell proliferation. The results together suggested that curcumin inhibited MCF-7 cell proliferation by inhibiting the assembly dynamics of microtubules.

• FtsZ inhibition: a promising approach for antistaphylococcal therapy.

  Authors: Parminder Singh, Dulal Panda

  Drug news & perspectives. 06/2010; 23(5):295-304.

  Staphylococcus causes a large number of animal and human diseases and has been considered as a major health concern. With the emergence of resistant strains of staphylococcus, likeStaphylococcus causes a large number of animal and human diseases and has been considered as a major health concern. With the emergence of resistant strains of staphylococcus, like methicillin-resistant Staphylococcus aureus and vancomycin-resistant Staphylococcus aureus, the search for novel antibacterial targets has intensified. FtsZ, a bacterial cytoskeleton protein, is involved in cell division. FtsZ assembles into protofilaments in a GTP-dependent manner, and forms a dynamic Z-ring at the mid-cell position. The assembly dynamics of FtsZ in the Z-ring are regulated by the combined actions of several FtsZ-associated proteins. Furthermore, the interaction of FtsZ with accessory proteins is essential for their recruitment to the Zring. A disruption of this interaction perturbs the Z-ring formation. FtsZ inhibitors like PC-190723 have been suggested to inhibit the Staphylococcus cell division by perturbing the assembly and stability of FtsZ polymers. In this review, we discuss the assembly dynamics of Z-ring and its role in cell division. In addition, we highlight recent advances suggesting the potential of FtsZ as a drug target for antistaphylococcal therapy.

• HMBA depolymerizes microtubules, activates mitotic checkpoints and induces mitotic block in MCF-7 cells by binding at the colchicine site in tubulin.

  Authors: Biswa Prasun Chatterji, Mithu Banerjee, Parminder Singh, Dulal Panda

  Biochemical pharmacology. 03/2010; 80(1):50-61.

  10-[(3-Hydroxy-4-methoxybenzylidene)]-9(10H)-anthracenone (HMBA), a synthetic compound, has been reported to have a potent antitumor activity. In this study, we found that HMBA depolymerized10-[(3-Hydroxy-4-methoxybenzylidene)]-9(10H)-anthracenone (HMBA), a synthetic compound, has been reported to have a potent antitumor activity. In this study, we found that HMBA depolymerized microtubules in MCF-7 cells and produced aberrant spindles in the MCF-7 cells. It also reduced the distance between the centrosomes and activated the mitotic checkpoint proteins BubR1 and Mad2. Further, HMBA inhibited the progression of MCF-7 cells in mitosis and induced apoptotic cell death involving p53 pathway. In vitro, HMBA bound to purified brain tubulin with a dissociation constant of 4.1+/-0.9 microM. It inhibited microtubule assembly and increased the GTP hydrolysis rate of microtubule assembly. The compound did not alter the binding of 2'(or 3')-O-(trinitrophenyl) guanosine 5'-triphosphate (TNP-GTP), a fluorescent analogue of GTP, to tubulin suggesting that it did not inhibit the binding of GTP to tubulin. However, we obtained evidence indicating that HMBA perturbed the conformation of the GTP binding site in tubulin. In addition, an analysis of the modified Dixon plot suggested that HMBA competitively inhibited the binding of colchicine to tubulin. A computational analysis of the binding of HMBA to tubulin supported the finding that HMBA shared its binding site with colchicine in tubulin and indicated that the binding of HMBA to tubulin was primarily stabilized through hydrogen bonding.

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

  Authors: Krishnan Rathinasamy, Bhavya Jindal, Jayant Asthana, Parminder Singh, Petety Balaji, Dulal Panda

  BMC Cancer. 01/2010;

  Abstract 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 lowAbstract 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 αβ 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.

• Fluorescence spectroscopic methods to analyze drug-tubulin interactions.

  Authors: Bhabatarak Bhattacharyya, Sonia Kapoor, Dulal Panda

  Methods in cell biology. 01/2010; 95:301-29.

  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,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.

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

  Authors: Krishnan Rathinasamy, Bhavya Jindal, Jayant Asthana, Parminder Singh, Petety V Balaji, Dulal Panda

  BMC cancer. 01/2010; 10:213.

  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, griseofulvinGriseofulvin, 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. 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. 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. 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.

• Targeting FtsZ for antibacterial therapy: a promising avenue.

  Authors: Sonia Kapoor, Dulal Panda

  Expert opinion on therapeutic targets. 10/2009; 13(9):1037-51.

  The widespread problem of bacterial resistance towards existing drugs and the paucity of effective drugs for the treatment of bacterial infections have prompted the scientific community to thinkThe widespread problem of bacterial resistance towards existing drugs and the paucity of effective drugs for the treatment of bacterial infections have prompted the scientific community to think about novel strategies for discovering new classes of antibacterial drugs. Target-based screening of inhibitory molecules has emerged as an important alternative for the development of potent antibacterials. FtsZ is a prokaryotic cytoskeleton protein, which plays an important role in bacterial cell division. It forms a highly dynamic Z-ring at the centre of the cell and recruits other accessory proteins, which are involved in bacterial cytokinesis. Here, we discuss the assembly dynamics of FtsZ and the key features that place it among the novel antibacterial drug targets. The recent progress in finding the inhibitors of functional properties of FtsZ and its interactions with other proteins, which has been enabled by advanced screening methods and structure-based design, are presented herein. Although there are significant challenges in the development of this new class of antibacterial drugs, nonetheless the therapeutic potential of FtsZ as a drug target is motivating researchers to find lead molecules with enhanced efficacy and reduced toxicity.

• Differential assembly properties of Escherichia coli FtsZ and Mycobacterium tuberculosis FtsZ: An analysis using divalent calcium.

  Authors: Richa Jaiswal, Dulal Panda

  Journal of biochemistry. 09/2009;

  The assembly of FtsZ is considered to be a fundamental process during the bacterial cytokinesis. We used several complimentary techniques to probe the assembly of recombinant Escherichia coli FtsZThe assembly of FtsZ is considered to be a fundamental process during the bacterial cytokinesis. We used several complimentary techniques to probe the assembly of recombinant Escherichia coli FtsZ (EcFtsZ) and Mycobacterium tuberculosis FtsZ (MtbFtsZ) proteins in vitro. As documented earlier, EcFtsZ was found to polymerize at much faster rate than MtbFtsZ. Interestingly, we found that MtbFtsZ produced higher sedimentable polymerized mass than that of the EcFtsZ and that MtbFtsZ formed thicker protofilaments than that of the EcFtsZ. The results indicated that the EcFtsZ polymers are more labile than the MtbFtsZ polymers. Further, divalent calcium exerted strikingly different effects on the assembly of EcFtsZ and MtbFtsZ. Divalent calcium strongly enhanced the assembly of EcFtsZ and promoted bundling and stability of the protofilaments. In contrast, it had no detectable effect on the assembly of MtbFtsZ. In vitro, divalent calcium bound to EcFtsZ with much stronger affinity than to MtbFtsZ and significantly affected the secondary structure of EcFtsZ whereas it did not cause any detectable change in the secondary structure of MtbFtsZ. The results suggested that the assembly characteristics of EcFtsZ and MtbFtsZ are different and indicated that the assembly dynamics of these proteins are regulated by different mechanisms.

• Promoting assembly and bundling of FtsZ as a strategy to inhibit bacterial cell division: a new approach for developing novel antibacterial drugs.

  Authors: Tushar Beuria, Parminder Singh, Avadhesha Surolia, Dulal Panda

  The Biochemical journal. 08/2009;

  FtsZ plays an essential role in bacterial cell division. We have used the assembly of FtsZ as a screen to find antibacterial agents with a novel mechanism of action. The effects of 81 compounds of 29FtsZ plays an essential role in bacterial cell division. We have used the assembly of FtsZ as a screen to find antibacterial agents with a novel mechanism of action. The effects of 81 compounds of 29 different structural scaffolds on FtsZ assembly in vitro were examined using a sedimentation assay. Out of these 81 compounds, 3-{5-[4-Oxo-2-thioxo-3-(3-trifluoromethyl-phenyl)-thiazolidin-5-ylidenemethyl]-furan-2-yl}-benzoic acid (OTBA) was found to promote FtsZ assembly in vitro. OTBA increased the assembly of FtsZ, caused bundling of FtsZ protofilaments, prevented dilution-induced disassembly of FtsZ protofilaments and decreased the GTPase activity in vitro. It bound to FtsZ with an apparent dissociation constant of 15 +/- 1.5 microM. Further, OTBA inhibited the proliferation of Bacillus subtilis 168 cells with a MIC of 2 microM while it exerted minimal effects on mammalian cell proliferation indicating that it might have a potential use as an antibacterial drug. In the effective proliferation inhibitory concentration range, OTBA induced filamentation in bacteria and also perturbed the formation of the cytokinetic Z-rings in bacteria. However, the agent neither perturbed the membrane structures nor affected the nucleoid segregation in B. subtilis cells. The results suggested that the OTBA inhibited bacterial cytokinesis by perturbing the formation and functioning of the Z-ring via altering FtsZ assembly dynamics. The antibacterial mechanism of action of OTBA is similar to that of the widely used anticancer drug paclitaxel, which inhibits cancer cell proliferation by promoting the assembly of tubulin, a eukaryotic homologue of FtsZ.