Tejashree Mahaddalkar

Structural investigations into the binding mode of a novel noscapine analogue, 9-(4-vinylphenyl) noscapine, with tubulin by biochemical analyses and molecular dynamic simulations.

Structural investigations into the binding mode of a novel noscapine analogue, 9-(4-vinylphenyl) noscapine, with tubulin by biochemical analyses and molecular dynamic simulations Tejashree Mahaddalkar, Pradeep Kumar Naik, Sinjan Choudhary, Naresh Manchukonda, Srinivas Kantevari and Manu Lopus* Experimental Cancer Therapeutics and Chemical Biology, UM-DAE Centre for Excellence in Basic Sciences, University of Mumbai Kalina Campus, Santacruz (E), Mumbai 400098, India; Department of Biotechnology, Guru Ghasidas Central University, Bilaspur, Chattisgarh 495009, India; Organic Chemistry Division-II (CPC Division), CSIR-Indian Institute of Chemical Technology, Hyderabad, Telangana 500007, India

Induction of acetylation and bundling of cellular microtubules by 9-(4-vinylphenyl) noscapine elicits S-phase arrest in MDA-MB-231 cells

Noscapine is an alkaloid present in the latex of Papaver somniferum. It has been known for its anticancer efficacy and lack of severe toxicities to normal tissues. Structural alterations in noscapine core architecture have produced a number of potent analogues of noscapine. Here, we report an unusual activity of a novel noscapine analogue, 9-(4-vinylphenyl)noscapine (VinPhe-Nos) on cancer cells. As we reported earlier, VinPhe-Nos inhibited MDA-MB-231 cell proliferation with an IC50 of 6μM. The present study elucidated a possible antiproliferative mechanism of action of VinPhe-Nos. The noscapinoid significantly inhibited clonogenic propagation of MDA-MB-231 cells. However, unlike the majority of tubulin-binding agents, it did not induce mitotic arrest; instead, it prolonged S-phase. Although prolonged presence of the drug show some disruption of cellular microtubule architecture, it did not affect microtubule recovery after cold-induced depolymerization. VinPhe-Nos, nevertheless, induced acetylation and bundling of microtubules. Our data suggest that rational modification of parent compound can alter its mechanism of action on cell cycle and that VinPhe-Nos can be investigated further as a less-toxic, S-phase-preferred, cytostatic anticancer agent.

Tryptone-stabilized gold nanoparticles target tubulin and inhibit cell viability by inducing an unusual form of cell cycle arrest

Abstract Gold nanoparticles have been investigated extensively for their molecular mechanisms of action and anticancer potential. We report a novel, tubulin‐targeted antiproliferative mechanism of action of tryptone‐stabilized gold nanoparticles (TsAuNPs). TsAuNPs, synthesized using HAuCl4·3H2O and tryptone and characterized by a variety of spectroscopic methods and transmission electron microscopy, were found to be inhibitory to viability of human pancreatic (PANC‐1), cervical (HeLa), and breast (MDA‐MB‐231) cancer cell lines in a concentration‐dependent manner, with highest efficacy against PANC‐1 cells. The particles strongly inhibited the clonogenic propagation of PANC‐1 cells. TsAuNPs‐mediated inhibition of cell viability involved an unusual mode of cell cycle arrest (arrest at both G0/G1 phase and S‐phase) followed by apoptosis. In vitro, TsAuNPs bound purified tubulin, competitively inhibited anilinonaphthalene sulfonate binding to tubulin, and suppressed tubulin assembly. In cells, tubulin‐TsAuNPs interactions were manifested as a disrupted microtubule network, defective reassembly of cold‐disassembled microtubules, and induction of tubulin acetylation. Our data indicate that TsAuNPs inhibit cell viability by inducing differential cell cycle arrest possibly through disrupted dynamicity of cellular microtubules. Graphical abstract Figure. No caption available. HighlightsTsAuNPs were synthesized using HAuCl4·3H2O and tryptone.TsAuNPs bound tubulin, promoted its acetylation, and inhibited its reassembly in cells.TsAuNPs induced G0/G1 and S‐phase arrests.The cells, thus arrested, eventually underwent programmed cell death.Combining TsAuNPs with a G2/M blocker might be a potential cancer therapeutic strategy.

Chemical synthesis, pharmacological evaluation and in silico analysis of new 2,3,3a,4,5,6-hexahydrocyclopenta[c]pyrazole derivatives as potential anti-mitotic agents

We have synthesized new, biologically active mono- and di-substituted 2,3,3a,4,5,6-hexahydrocyclopenta[c]pyrazole derivatives bearing electron withdrawing groups and electron donating groups. These derivative structures were characterized by their spectral and analytical data. The newly synthesized hexahydropyrazole analogues were evaluated for their in vitro anticancer activity against breast and lung cancer cell lines using a cytotoxicity bioassay. To understand their mechanism of action, tubulin binding assays were performed which pointed to their binding to microtubules in a mode similar to but not identical to colchicine, as evidenced by their KD value evaluation. Computational docking studies also suggested binding near the colchicine binding site on tubulin. These results were further confirmed by colchicine-binding assays on the most active compounds, which indicated that they bound to tubulin near but not at the colchicine site. The moderate cytotoxic effects of these compounds may be due to the presence of electron donating groups on the para-position of the phenyl ring, along with the hexahydropyrazole core nucleus. The observed anti-cancer activity based on inhibition of microtubule formation may be helpful in designing more potent compounds with a hexahydropyrazole moiety.

Aqueous extract of Triphala inhibits cancer cell proliferation through perturbation of microtubule assembly dynamics

Triphala (Trl) is an ayurvedic formulation used for treating disorders of the digestive, respiratory, and nervous systems. Its anticancer properties have also been documented. We studied effects of Trl on tubulin, a target protein for several anticancer drugs, and systematically elucidated a possible antiproliferative mechanism of action of Trl. Trl inhibited proliferation of HeLa (cervical adenocarcinoma), PANC-1 (pancreatic adenocarcinoma), and MDA-MB-231 (triple-negative breast carcinoma) cells in microgram quantities and strongly suppressed the clonogenicity of HeLa cells. The formulation disrupted secondary conformation of tubulin and inhibited anilino naphthalene sulfonate binding to tubulin. In cells, Trl-tubulin interactions were manifested as a perturbed microtubule network. Acetylation pattern of Trl-treated cellular microtubules indicated persistent stabilization of microtubule dynamics. In addition, Trl interfered with reassembly of the microtubules. Cells treated with Trl eventually underwent programmed cell death as evidenced by annexin-V staining. Our study shows that the effect of aqueous extract of Trl is potent enough to interfere with the assembly dynamics of microtubules, and that Trl can be investigated further for its antitumor potential.

Elucidation of the Tubulin-targeted Mechanism of Action of 9-(3-pyridyl) Noscapine

We have recently reported the synthesis and antiproliferative potential of a series of biaryl type α-noscapine congeners. Among them, 9-(3-pyridyl) noscapine 3f (9-PyNos, henceforth), which was synthesized by adding pyridine unit to the tetrahydroisoquinoline part of natural α-noscapine core, was found to be the most effective one to inhibit proliferation of a variety of cancer cell lines. However, details of its interactions with its cellular target, tubulin, remain poorly understood. In this report, we examined the nature of interactions of 9-PyNos with tubulin based on the methodologies of spectrofluorimetry, circular dichroism, and turbidimetry techniques. Far-UV circular dichroism spectra indicated perturbation of tubulin secondary structure in the presence of 9-PyNos, not amounting, however, to the perturbation induced by noscapine. The noscapinoid nevertheless altered the surface configuration of the protein considerably, as indicated by an anilinonaphthalene sulphonate binding assay, and promoted colchicine binding to tubulin, the latter indicating its adjacent binding site with colchicine. 9-PyNos however, did not alter microtubule assembly considerably. Investigating the possible reason behind this apparent lack of strong inhibition of microtubule assembly, we found that the binding interactions of tubulin with 9-PyNos do not involve modification of cysteine residues of tubulin. Taken together, our data suggest that the antiproliferative mechanism of action of 9-PyNos involves disruption of structural integrity of tubulin without strong inhibition of tubulin assembly.