title: Mechanism of action of the antimitotic drug 2,4-dichlorobenzyl thiocyanate: alkylation of sulfhydryl group(s) of beta-tubulin.
abstract: The compound, 2,4-dichlorobenzyl thiocyanate (DCBT) was previously shown to cause mitotic arrest, disruption of intracellular microtubules, and inhibition of tubulin polymerization, with resistance to the drug conferred by a mutation in a beta-tubulin gene (Abraham, I., Dion, R.L., Duanmu, C., Gottesman, M.M. and Hamel, E. (1986) Proc. Natl. Acad. Sci. USA 83, 6839-6843). We have now examined its mechanism of action in further detail and conclude that DCBT acts as a sulfhydryl alkylating reagent. A mixed disulfide forms between the 2,4-dichlorobenzyl mercaptan moiety of DCBT and protein sulfhydryl groups with release of cyanate anion to the medium. Gel filtration and dialysis of complexes of tubulin formed with either [nitrile-14C]DCBT, [35S]DCBT or [benzyl-3H]DCBT demonstrated persistent association of 35S and 3H with denatured tubulin, but no binding of 14C to the protein even under native conditions. With equimolar tubulin and DCBT, beta-tubulin is the predominant alkylated species. At high drug concentrations, superstoichiometric amounts of DCBT react with tubulin, and both subunits are alkylated almost equally. When extracts of drug-treated L1210 murine leukemia cells were examined by polyacrylamide gel electroporesis, we found that multiple proteins were alkylated by DCBT, but the most prominent radiolabeled band was that corresponding to beta-tubulin. Dithiothreitol partially reverses inhibition of tubulin polymerization by DCBT and removes almost all the 2,4-dichlorobenzyl mercaptan moiety covalently bound to tubulin. Mitotic arrest occurs with DCBT because tubulin is the cellular protein most sensitive to the agent, probably because of its high cysteine content (20/mol).
authors.affiliation: Laboratory of Pharmacology and Experimental Therapeutics, National Cancer Institute, Bethesda, MD 20892.
authors.forename: R
authors.initials: R
authors.lastname: Bai
authors.forename: C
authors.initials: C
authors.lastname: Duanmu
authors.forename: E
authors.initials: E
authors.lastname: Hamel
mesh_terms: D000478:Alkylation; D000818:Animals; D000970:Antineoplastic Agents; D002250:Carbon Radioisotopes; D002417:Cattle; D002850:Chromatography, Gel; D003485:Cyanates / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D007939:Leukemia L1210 / Q000378:metabolism; D051379:Mice; D011489:Protein Denaturation; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013462:Sulfur Radioisotopes; D013861:Thiocyanates / Q000378:metabolism / Q000494:pharmacology*; D014316:Tritium; D014404:Tubulin / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; D002250:Carbon Radioisotopes; D003485:Cyanates; D004220:Disulfides; D013438:Sulfhydryl Compounds; D013462:Sulfur Radioisotopes; D013861:Thiocyanates; D014404:Tubulin; D014316:Tritium; C050043:2,4-dichlorobenzyl thiocyanate; D004229:Dithiothreitol