P450 inhibitors (Bateman et al., 2004; Blobaum et al., 2002; Correia and Ortiz
de Montellano, 2005; Koenigs et al., 1999). A potentially higher success rate is
expected in protein adduct characterization by applying the modern proteomic
approaches such as tryptic digestion followed by multidimensional separation
and high resolution mass spectrometry (Zhou, 2003). However, a few problems
intrinsic to protein adduct identification cannot be solved by just the
advancement of analytical techniques. These problems include the specific
difficulty in proteomic research of membrane proteins such as cytochrome P450
enzymes, instability of some drug–protein adducts, and the lack of specificity of
modification sites. Direct mass measurement by MALDI–TOF may circumvent
the adduct instability problem, but no detailed information on modification site
of proteins can be obtained. Because of the mechanistic nature of these studies
and the resources and expertise requirement, experimental protocols for
proteomic research will need some time to be optimized.
In addition to reactive intermediates formed in Phase I metabolism, some
metabolites (e.g., acyl glucuronides) resulted from Phase II metabolism might
also react with proteins to form corresponding adducts, which might result in
observed drug-related adverse effects. Acyl glucuronides and xenobiotic acyl
thioesters are two important classes of electrophilic intermediates (Boelsterli,
2002). Two major mechanisms have been proposed for the reactions of acyl
glucuronides with proteins. The first mechanism is a nucleophilic acyl
substitution, resulting in acylation of proteins. The second is involved in a
glycation via chain open acyl migration processes (Boelsterli, 2002; Ritter,
2000; Shipkova et al., 2003). Recently, a few groups have shown interesting
approaches for the screening of adduct formation from acyl glucuronides
(Bolze et al., 2002; Wang et al., 2004). One of these approaches is to use a
synthetic lysine-containing peptide to react with acyl glucuronide to form a
Schiff’s base that can be assayed by LC/MS/MS, and the relative amount of
peptide adduct formation may be correlated to the relative amount of protein
adduct formation by a variety of acyl glucuronides. This approach requires use
of acyl glucuronides, which may be obtained either via purification from
incubations within vitro metabolism systems or from chemical synthesis.
Alternatively, investigators may consider performingin vitroinstability studies
of acyl glucuronides to assess their potential liability as reactive intermediates
(Wen et al., 2006). In addition, acyl glucuronide metabolites may also react
with DNA to cause potential genotoxicity. It was reported that the acyl
glucuronides of two widely used fibrate hypolipidemics, clofibric acid, and
gemfibrozil, caused a concentration-dependent decrease in the transfection
efficiency of the DNA inEscherichia Coli, with a greater than 80% decrease in
phage survival in the presence of 5 mM glucuronides (Sallustio et al., 1997). It
was also demonstrated that the acyl glucuronides of probenecid and clofibric
may have induced DNA damage in isolated hepatocytes (Sallustio et al., 2006).
The mechanism for the acyl CoA thioesters involves in the reaction with
proteins through nucleophilic acyl substitution. It was reported that the acyl
CoA-thioester of zomepirac, together with the acyl glucuronide of zomepirac,
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