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C‐12 Binds to Tubulin at the Colchicine‐binding Site and Target Microtubules by Preferentially Binding to GTP‐bound Tubulin
Author(s) -
Prassanawar Shweta Shyam,
Kumari Anuradha,
Guchhait Sankar K,
Panda Dulal
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.784.14
Subject(s) - tubulin , microtubule , colchicine , mitosis , chemistry , gtp' , binding site , microbiology and biotechnology , biology , biochemistry , genetics , enzyme
In eukaryotic cells, microtubules are essential for cell division as they assemble into dynamic mitotic spindles which sequester and segregate the duplicated chromosomes. Microtubule‐targeting agents (MTAs) inhibit the dynamicity of the microtubules, thereby hampering chromosome segregation and ultimately inhibiting cancer cell proliferation. A natural compound combretastatin A‐4 (CA‐4), a potent MTA, is undergoing several clinical trials for the treatment of various cancers. However, its toxicity and low bioavailability present a major challenge for it to be considered as an approved drug. C‐12, a derivative of CA‐4, has been found to be more potent than CA‐4 in inhibiting proliferation of several cancer cell lines. In this work, we examined the binding characteristics of C‐12 and its effect in triple negative breast cancer (TNBC) cells. C‐12 inhibited the assembly of microtubules, in vitro , as observed by a turbidimetry assay and electron microscopy. A molecular docking study suggested that C‐12 interacts with tubulin with a binding energy of −10.53 kcal/mol and prefers the colchicine‐binding site on the tubulin dimer. This was further confirmed in a competitive assay, where podophyllotoxin, a colchicine site binder, strongly inhibited the binding of C‐12 to tubulin with an inhibitory constant (K i ) of 0.6 ± 0.2 μM. The fluorescence intensity of C‐12 increases upon binding to tubulin. Therefore, we analyzed the binding of C‐12 to tubulin using the fluorescence of C‐12. Interestingly, the compound exhibited a relatively greater affinity for GTP‐bound tubulin than the GDP‐bound tubulin. The dissociation constant (K d ) for C‐12 and GTP‐tubulin interaction was found to be 1 ± 0.8 μM which is significantly less than the K d of C‐12 and GDP‐tubulin (9.2 ± 1.2 μM). This suggested that in cells, C‐12 might preferentially bind to the growing plus end of the microtubules. We also examined the effects of C‐12 on the cultured TNBC cells. Immunofluorescence analysis revealed that C‐12 depolymerized the interphase microtubule network and perturbed the localization of γ‐tubulin in TNBC cells. Moreover, the compound hampered chromosome congression and blocked the cells at mitosis, thereby inhibiting cell division. The results, altogether suggested that C‐12 is a potent microtubule targeting agent that binds to tubulin at the colchicine site and perturbs microtubule function in cells. Support or Funding Information This work is supported by TATA Innovation fellowship awarded to D.P. from Department of Biotechnology, Government of India. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .