NMK-TD-100, a novel microtubule modulating agent, blocks mitosis and induces apoptosis in HeLa cells by binding to tubulin

PLoS One. 2013 Oct 7;8(10):e76286. doi: 10.1371/journal.pone.0076286. eCollection 2013.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acids / chemistry
  • Amino Acids / metabolism
  • Apoptosis / drug effects*
  • Blotting, Western
  • Caspase 3 / metabolism
  • Cell Proliferation / drug effects
  • Cell Survival / drug effects
  • Cells, Cultured
  • Dose-Response Relationship, Drug
  • HeLa Cells
  • Humans
  • Indoles / chemistry
  • Indoles / metabolism
  • Indoles / pharmacology
  • Leukocytes, Mononuclear / cytology
  • Leukocytes, Mononuclear / drug effects
  • Leukocytes, Mononuclear / metabolism
  • M Phase Cell Cycle Checkpoints / drug effects
  • Membrane Potential, Mitochondrial / drug effects
  • Microscopy, Electron
  • Microtubules / drug effects
  • Microtubules / metabolism
  • Mitosis / drug effects*
  • Models, Molecular
  • Molecular Structure
  • Protein Binding
  • Protein Structure, Tertiary
  • Proto-Oncogene Proteins c-bcl-2 / metabolism
  • Thiadiazoles / chemistry
  • Thiadiazoles / metabolism
  • Thiadiazoles / pharmacology
  • Tubulin / chemistry
  • Tubulin / metabolism*
  • Tubulin / ultrastructure
  • Tubulin Modulators / chemistry
  • Tubulin Modulators / metabolism
  • Tubulin Modulators / pharmacology*
  • Tumor Suppressor Protein p53 / metabolism

Substances

  • Amino Acids
  • Indoles
  • NMK-TD-100
  • Proto-Oncogene Proteins c-bcl-2
  • Thiadiazoles
  • Tubulin
  • Tubulin Modulators
  • Tumor Suppressor Protein p53
  • Caspase 3

Grants and funding

The work was supported by grant from DBT, Govt. of India (No. BT/PR12889/AGR/36/624/2009) to GC. SB is a Senior Research fellow of University Grants Commission (UGC), Govt. of India. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.