Inhibition of tubulin assembly and covalent binding to microtubular protein by valproic acid glucuronide in vitro

Cannell, GR, Bailey, MJ and Dickinson, RG (2002) Inhibition of tubulin assembly and covalent binding to microtubular protein by valproic acid glucuronide in vitro. Life Sciences, 71 22: 2633-2643. doi:10.1016/S0024-3205(02)02107-0

Author Cannell, GR
Bailey, MJ
Dickinson, RG
Title Inhibition of tubulin assembly and covalent binding to microtubular protein by valproic acid glucuronide in vitro
Journal name Life Sciences   Check publisher's open access policy
ISSN 0024-3205
Publication date 2002-01-01
Sub-type Article (original research)
DOI 10.1016/S0024-3205(02)02107-0
Open Access Status Not yet assessed
Volume 71
Issue 22
Start page 2633
End page 2643
Total pages 11
Editor H. I. Yamamura
Place of publication New York
Publisher Elsevier Science
Language eng
Subject C1
320504 Toxicology (incl. Clinical Toxicology)
730199 Clinical health not specific to particular organs, diseases and conditions
Abstract Acyl glucuronides are reactive metabolites of carboxylate drugs, able to undergo a number of reactions in vitro and in vivo, including isomerization via intramolecular rearrangement and covalent adduct formation with proteins. The intrinsic reactivity of a particular acyl glucuronide depends upon the chemical makeup of the drug moiety. The least reactive acyl glucuronide yet reported is valproic acid acyl glucuronide (VPA-G), which is the major metabolite of the antiepileptic agent valproic acid (VPA). In this study, we showed that both VPA-G and its rearrangement isomers (iso-VPA-G) interacted with bovine brain microtubular protein (MTP, comprised of 85% tubulin and 15% microtubule associated proteins {MAPs}). MTP was incubated with VPA, VPA-G and iso-VPA-G for 2 h at room temperature and pH 7.5 at various concentrations up to 4 mM. VPA-G and iso-VPA-G caused dose-dependent inhibition of assembly of MTP into microtubules, with 50% inhibition (IC50) values of 1.0 and 0.2 mM respectively, suggesting that iso-VPA-G has five times more inhibitory potential than VPA-G. VPA itself did not inhibit microtubule formation except at very high concentrations (greater than or equal to2 mM). Dialysis to remove unbound VPA-G and iso-VPA-G (prior to the assembly assay) diminished inhibition while not removing it. Comparison of covalent binding of VPA-G and iso-VPA-G (using [C-14]-labelled species) showed that adduct formation was much greater for iso-vTA-G. When [C-14]-iso-VPA-G was reacted with MTP in the presence of sodium cyanide (to stabilize glycation adducts), subsequent separation into tubulin and MAPs fractions by ion exchange chromatography revealed that 78 and 22% of the covalent binding occurred with the MAPs and tubulin fractions respectively. These experiments support the notion of both covalent and reversible binding playing parts in the inhibition of microtubule formation from MTP (though the acyl glucuronide of VPA is less important than its rearrangement isomers in this regard), and that both tubulin and (perhaps more importantly) MAPs form adducts with acyl glucuronides. (C) 2002 Elsevier Science Inc. All rights reserved.
Keyword Medicine, Research & Experimental
Pharmacology & Pharmacy
Valproic Acid
Drug-protein Adducts
Diflunisal Acyl Glucuronide
Tandem Mass-spectrometry
Dipeptidyl Peptidase-iv
Human Serum-albumin
Irreversible Binding
Resistant Glucuronides
Adduct Formation
Q-Index Code C1
Institutional Status Unknown

Document type: Journal Article
Sub-type: Article (original research)
Collections: Excellence in Research Australia (ERA) - Collection
School of Medicine Publications
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Citation counts: TR Web of Science Citation Count  Cited 22 times in Thomson Reuters Web of Science Article | Citations
Scopus Citation Count Cited 25 times in Scopus Article | Citations
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Created: Wed, 15 Aug 2007, 04:00:36 EST