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

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Li CM, Lu Y, Ahn S, Narayanan R, Miller DD, Dalton JT

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

J Mass Spectrom. 2010 Oct;45(10):1160-6. doi: 10.1002/jms.1804.

PubMed ID

20814887 [ View in PubMed

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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 tandem mass 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.

DrugBank Data that Cites this Article

Drug Targets

Drug	Target	Kind	Organism	Pharmacological Action	Actions
Colchicine	Tubulin beta chain	Protein	Humans	Yes	Inhibitor Binder	Details
Podofilox	Tubulin alpha-4A chain	Protein	Humans	Yes	Inhibitor	Details