Competitive mass spectrometry binding assay for characterization of three binding sites of tubulin

Chien Ming Li, Yan Lu, Sunjoo Ahn, Ramesh Narayanan, Duane Miller, James T. Dalton

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

Tubulin is an attractive and established target for anticancer therapy. To date, the only method to determine the binding of inhibitor to tubulin has been competitive radioligand binding assays. We developed a non-radioactive mass spectrometry (MS) binding assay to study the tubulin binding of colchicine, vinblastine and paclitaxel and to identify which of these three binding sites that a novel inhibitor binds. The method involves a very simple step of separating the unbound ligand from macromolecules using ultrafiltration. The unbound ligand in the filtrate can be accurately determined using highly sensitive and specific liquid chromatography tandemmass spectrometry (LC-MS/MS)method using multiple reaction monitoring (MRM) mode. The assay was validated using podophyllotoxin, vincristine and docetaxel, drugs that compete to the colchicine-, vinblastine- and paclitaxel-binding sites in tubulin, respectively. This competitive binding assay allowed the reliable detection of interactions of these drugs with three binding sites on tubulin. This method was subsequently applied to determine the tubulin-binding site of 4-substituted methoxylbenzoyl-aryl-thiazoles (SMART-H), a potent antitubulin agent developed in our laboratory. The results indicated that SMART-H specifically and reversibly bound only to the colchicine-binding site, but not to vinblastine- or paclitaxel sites. This new non-radioligand binding method to determine the binding site on tubulin will function as a useful tool to study the binding sites of tubulin inhibitors.

Original languageEnglish (US)
Pages (from-to)1160-1166
Number of pages7
JournalJournal of Mass Spectrometry
Volume45
Issue number10
DOIs
StatePublished - Oct 1 2010

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Tubulin
Mass spectrometry
Assays
Binding Sites
Vinblastine
Colchicine
Paclitaxel
docetaxel
Tubulin Modulators
Podophyllotoxin
Ligands
Thiazoles
Liquid chromatography
Vincristine
Ultrafiltration
Macromolecules
Pharmaceutical Preparations
Spectrometry
Monitoring

All Science Journal Classification (ASJC) codes

  • Spectroscopy

Cite this

Competitive mass spectrometry binding assay for characterization of three binding sites of tubulin. / Li, Chien Ming; Lu, Yan; Ahn, Sunjoo; Narayanan, Ramesh; Miller, Duane; Dalton, James T.

In: Journal of Mass Spectrometry, Vol. 45, No. 10, 01.10.2010, p. 1160-1166.

Research output: Contribution to journalArticle

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