ZOMEPIRAC ACYL GLUCURONIDE COVALENTLY MODIFIES TUBULIN IN-VITRO AND IN-VIVO AND INHIBITS ITS ASSEMBLY IN AN IN-VITRO SYSTEM

Drugs possessing a carboxylate functional group usually form acyl gluc uronides as major metabolites. These electrophilic metabolites can und ergo several spontaneous reactions, including covalent adduct formatio n with proteins. The present study examined whether covalent adducts w ere formed with microtubular protein (MTP, 85% alpha/beta-tubulin) and whether this influenced its ability to assemble into microtubules. Bo vine brain microtubular protein (MTP) was purified by assembly-disasse mbly cycles and incubated with the nonsteroidal anti-inflammatory drug (NSAID) zomepirac (ZP), its acyl glucuronide (ZAG) and rearrangement isomers (iso-ZAG) at various concentrations for 2 h at room temperatur e and pH 7.5. Assembly was monitored by change in turbidity (increase in absorbance at 340 nm). Both ZAG and iso-ZAG caused dose-dependent i nhibition of assembly (50% inhibition at about 1 mM), while ZP caused modest inhibition (< 50% inhibition at 4 mM). In a slightly different system, incubation of performed microtubules with 4 mM ZAG caused abou t 35% inhibition of reassembly ability, while modification of MTP unde r similar conditions resulted in about 85% reduction of assembly abili ty. Immunoblotting with a ZP antiserum showed that ZAG and iso-ZAG cov alently modified MTP in a dose-dependent manner, while ZP itself cause d no modification. Tubulin and many minor proteins comprising MTP were modified. ZP-modified tubulin was shown to be present in the cytosol of livers from rats dosed twice daily for 3 days with ZP at 50 mg/kg, using a sandwich ELISA with ZP and tubulin antisera. Whether any pertu rbation of microtubule assembly occurs in vivo as a result of this in vivo modification is currently under investigation. (C) 1998 Published by Elsevier Science Ireland Ltd. All rights reserved.