FOR COVALENT BINDING OF BENOXAPROFEN GLUCURONIDE TO HUMAN SERUM ALBUMIN Studies by Tandem Mass Spectrometry

Tandem MS has been used to establish the structure and specific binding sites of covalent protein adducts formed upon incubation of the acyl glucuronide of the propionic acid nonsteroidal antiinflammatory drug benoxaprofen with human serum albumin in vitro. Benoxaprofen 1-O-b-glucuronide was enzymatically synthesized in vitro and incubated with human serum albumin both in the presence and in the absence of NaCNBH3. The modified human serum albumins were digested with trypsin and separated by HPLC. The modified peptides were detected using HPLC-electrospray MS (with selected-ion monitoring) and were structurally characterized by tandem MS using matrix-assisted laser desorption ionization in both the post-source decay and high-energy collision-induced dissociation modes. These studies established that benoxaprofen glucuronide forms covalent adducts with protein nucleophiles both by nucleophilic displacement of glucuronic acid at the anomeric center and by condensation of the rearranged acyl glucuronic acid isomers with e-amino functions of lysine residues after acyl migration of the aglycone from the anomeric center. Lys-159 was identified as the major binding site. Thus, we have established that members of the less reactive propionic acid class of acyl glucuronides, such as the glucuronide of benoxaprofen, are also capable of reacting with protein nucleophiles to form covalent adducts analogous to those of tolmetin glucuronide (tolmetin is an acetic acid nonsteroidal anti-inflammatory drug), via the mechanisms previously reported from this laboratory, and that the specific covalent binding site profile appears to be drug dependent. The formation of acyl glucuronides is a major metabolic pathway for many compounds containing a carboxylic acid function, especially NSAIDs. These acyl glucuronides can undergo hydrolysis as well as spontaneous isomerization from the anomeric center at physiological pH and are capable of reacting with protein nucleophiles, to form covalent adducts, either directly or after isomerization and tautomerism (Dinget al., 1995; Faed, 1984). Some of these drugs cause toxic effects such as hypersensitivity (Zia-Amirhosseini et al., 1995) and idiosyncratic liver toxicity (Boelsterli et al., 1995), raising the question of whether covalent modification of proteins could play an important role in initiating these effects. Several recent studies have been carried out to establish the mechanisms for formation of covalent protein adducts with this class of drug metabolites. One mechanism involves nucleophilic displacement of the glucuronosyl group by -NH2 (van Breemenet al., 1986; McDonaghet al., 1984), -SH (van Breemen and Fenselau, 1985), or -OH (Wells t al., 1987; Rueliuset al., 1986) groups of the protein, to form covalent drug-protein adducts devoid of the glucuronic acid group. Another mechanism (Smith et al., 1986, 1990) involves the spontaneous migration of the acyl group to the 2-, 3-, or 4-hydroxyl groups of the sugar moiety, tautomerization of the pyranose ring to its aldose form, and condensation of the aldehyde group of the ring-opened tautomer with a lysine e-amino on the protein to form an imine. Stabilization of such an imine adduct would be expected to occur slowly in vivo, by spontaneous Amadori rearrangement to the more stable 1-amino-1-deoxy-ketose analogue (Hodge, 1955). However, the reversibly formed imine can be converted to the corresponding secondary amine in the presence of NaCNBH3, thus increasing the chemical stability and yield for structural analysis (Dinget al., 1995; Smithet al., 1990; Munafoet al., 1990; Dinget al., 1993). Previous contributions from this laboratory have reported the structural nature and extent of adduct formation of TG (tolmetin is an acetic acid NSAID) with HSA, both in the presence and in the absence of an imine-reducing agent (Ding et al., 1993, 1995). Those studies established that protein adducts are formed by both mechanisms and that Lys-199 is the major binding site for both adducts, together with a number of less prominent binding sites. Recently, we extended our studies to include the acyl glucuronides of propionic acid NSAIDs, which are much less chemically reactive than are those of acetic acid NSAIDs, because of steric hindrance and electron-donating effects of the a-methyl group. Now we report results on the reactivity of benoxaprofen [2-( p-chlorophenyl)a-methyl-5-benzoxazoleacetic acid] with HSA. Financial support was provided by National Institutes of Health National Center for Research Resources Grant 01614 (to A.L.B.), Grants DK26743 (to A.L.B.) and GM36633 (to L.Z.B.). 1 Abbreviations used are: NSAID, nonsteroidal anti-inflammatory drug; BG, benoxaprofen-1-O-b-glucuronide; SIM, selected-ion monitoring; CID, collisioninduced dissociation; PSD, post-source decay; HSA, human serum albumin; MALDI, matrix-assisted laser desorption ionization; TOF, time of flight; TG, tol-