title: NMK-TD-100, a novel microtubule modulating agent, blocks mitosis and induces apoptosis in HeLa cells by binding to tubulin.
abstract: Thiadiazoles are one of the most widely utilized agents in medicinal chemistry, having a wide range of pharmacologic activity. Microtubules (MTs) have always remained a sought-after target in rapidly proliferating cancer cells. We screened for the growth inhibitory effect of synthetic 5-(3-indolyl)-2-substituted-1,3,4-thiadiazoles on cancer cells and identified NMK-TD-100, as the most potent agent. Cell viability experiments using human cervical carcinoma cell line (HeLa cells) indicated that the IC50 value was 1.42±0.11 µM for NMK-TD-100 for 48 h treatment. In further study, we examined the mode of interaction of NMK-TD-100 with tubulin and unraveled the cellular mechanism responsible for its anti-tumor activity. NMK-TD-100 induced arrest in mitotic phase of cell cycle, caused decline in mitochondrial membrane potential and induced apoptosis in HeLa cells. Immunofluorescence studies using an anti-α-tubulin antibody showed a significant depolymerization of the interphase microtubule network and spindle microtubule in HeLa cells in a concentration-dependent manner. However, the cytotoxicity of NMK-TD-100 towards human peripheral blood mononuclear cells (PBMC) was lower compared to that in cancer cells. Polymerization of tissue purified tubulin into microtubules was inhibited by NMK-TD-100 with an IC50 value of 17.5±0.35 µM. The binding of NMK-TD-100 with tubulin was studied using NMK-TD-100 fluorescence enhancement and intrinsic tryptophan fluorescence of tubulin. The stoichiometry of NMK-TD-100 binding to tubulin is 1:1 (molar ratio) with a dissociation constant of ~1 µM. Fluorescence spectroscopic and molecular modeling data showed that NMK-TD-100 binds to tubulin at a site which is very near to the colchicine binding site. The binding of NMK-TD-100 to tubulin was estimated to be ~10 times faster than that of colchicine. The results indicated that NMK-TD-100 exerted anti-proliferative activity by disrupting microtubule functions through tubulin binding and provided insights into its potential of being a chemotherapeutic agent.
authors.affiliation: Department of Biotechnology and Dr. B.C. Guha Centre for Genetic Engineering and Biotechnology, University of Calcutta, Kolkata, India.
authors.forename: Surela
authors.initials: S
authors.lastname: Bhattacharya
authors.forename: N Maruthi
authors.initials: NM
authors.lastname: Kumar
authors.forename: Arnab
authors.initials: A
authors.lastname: Ganguli
authors.forename: Mukund P
authors.initials: MP
authors.lastname: Tantak
authors.forename: Dalip
authors.initials: D
authors.lastname: Kumar
authors.forename: Gopal
authors.initials: G
authors.lastname: Chakrabarti
mesh_terms: D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D006367:HeLa Cells; D006801:Humans; D007211:Indoles / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D059566:M Phase Cell Cycle Checkpoints / Q000187:drug effects; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008854:Microscopy, Electron; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism; D008938:Mitosis / Q000187:drug effects*; D008958:Models, Molecular; D015394:Molecular Structure; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D013830:Thiadiazoles / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D050257:Tubulin Modulators / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
chemical_list: D000596:Amino Acids; D007211:Indoles; C000590112:NMK-TD-100; D019253:Proto-Oncogene Proteins c-bcl-2; D013830:Thiadiazoles; D014404:Tubulin; D050257:Tubulin Modulators; D016159:Tumor Suppressor Protein p53; D053148:Caspase 3