Zomepirac acyl glucuronide covalently modifies tubulin in vitro and in vivo and inhibits its assembly in an in vitro system

Abstract

Drugs possessing a carboxylate functional group usually form acyl glucuronides as major metabolites. These electrophilic metabolites can undergo several spontaneous reactions, including covalent adduct formation with proteins. The present study examined whether covalent adducts were formed with microtubular protein (MTP, 85% α/ β-tubulin) and whether this influenced its ability to assemble into microtubules. Bovine brain microtubular protein (MTP) was purified by assembly-disassembly cycles and incubated with the non-steroidal anti-inflammatory drug (NSAID) zomepirac (ZP), its acyl glucuronide (ZAG) and rearrangement isomers (iso-ZAG) at various concentrations for 2 h at room temperature 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 inhibition 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 about 35% inhibition of reassembly ability, while modification of MTP under similar conditions resulted in about 85% reduction of assembly ability. Immunoblotting with a ZP antiserum showed that ZAG and iso-ZAG covalently modified MTP in a dose-dependent manner, while ZP itself caused 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 perturbation of microtubule assembly occurs in vivo as a result of this in vivo modification is currently under investigation.