Quantitative protein determination for CYP induction via LC-MS/MS.

The Cytochrome P450 (CYP) proteins are a family of membrane bound proteins that function as a major metabolizing enzyme in the human body. Quantification of CYP induction is critical in determining the disposition, safety and efficacy of drugs in humans. Described is a gel-free, high-throughput LC-MS approach to quantitate the CYP isoforms 1A2, 2B6, 3A4 and 3A5 by measuring isoform specific peptides released by enzymatic digestion of the hepatocyte incubations. The method uses synthetic stable isotope-labeled peptides as internal standards and allows both relative and absolute quantification to be performed from hepatic microsomal preparations. CYP protein determined by this LC-MS method correlated well with the mRNA and activity for induced levels of CYP1A2, CYP2B6 and CYP3A4. Interestingly, a small fold change was observed for the induction of 3A5 with phenobarbital. The results were reproducible with an average CV less then 10% for repeat analysis of the sample. This LC-MS method offers a robust assay for CYP protein quantitation for use in CYP induction assays.

Single mutations change CYP2F3 from a dehydrogenase of 3-methylindole to an oxygenase.

Pulmonary cytochrome P450 2F3 (CYP2F3) catalyzes the dehydrogenation of the pneumotoxin 3-methylindole (3MI) to an electrophilic intermediate, 3-methyleneindolenine, which is responsible for the toxicity of the parent compound. Members of the CYP2F subfamily are the only enzymes known to exclusively dehydrogenate 3MI, without detectable formation of oxygenation products. Thus, CYP2F3 is an attractive model to study dehydrogenation mechanisms. The purpose of this study was to identify specific residues that could facilitate 3MI dehydrogenation. Both single and double mutations were constructed to study the molecular mechanisms that direct dehydrogenation. Double mutations in substrate recognition sites (SRS) 1 produced an inactive enzyme, while double mutants in SRS 4 did not alter 3MI metabolism. However, double mutations in SRS 5 and SRS 6 successfully introduced oxygenase activity to CYP2F3. Single mutations in SRS 5, SRS 6 and near SRS 2 also introduced 3MI oxygenase activity. Mutants S474H and D361T oxygenated 3MI but also increased dehydrogenation rates, while G214L, E215Q and S475I catalyzed 3MI oxygenation exclusively. A homology model of CYP2F3 was precisely consistent with specific dehydrogenation of 3MI via initial hydrogen atom abstraction from the methyl group. In addition, intramolecular kinetic deuterium isotope studies demonstrated an isotope effect ( K H/ K D) of 6.8. This relatively high intramolecular deuterium isotope effect confirmed the initial hydrogen abstraction step; a mutant (D361T) that retained the dehydrogenation reaction exhibited the same deuterium isotope effect. The results showed that a single alteration, such as a serine to isoleucine change at residue 475, dramatically switched catalytic preference from dehydrogenation to oxygenation.

Probing adenosine-to-inosine editing reactions using RNA-containing nucleoside analogs.

Advances in chemical synthesis and characterization of nucleic acids allows for atom-specific modification of complex RNAs, such as present in RNA editing substrates. By preparing substrates for ADARs by chemical synthesis, it is possible to subtly alter the structure of the edited nucleotide. Evaluating the effect these changes have on the rate of enzyme-catalyzed deamination reveals features of the editing reaction and guides the design of inhibitors. We describe the synthesis of select nucleoside analog phosphoramidites and their incorporation into RNAs that mimic known editing sites by solid phase synthesis, and analyze the interaction of these synthetic RNAs with ADARs using deamination kinetics and quantitative gel mobility shift assays.

Metabolism and bioactivation of 3-methylindole by human liver microsomes.

Metabolism and bioactivation of 3-methylindole (3MI) were investigated in human liver microsomes. The metabolism of two deuterium-labeled analogues of 3MI permitted a relatively unambiguous identification of multiple metabolites and glutathione (GSH) adducts of reactive intermediates. A total of eight oxidized metabolites were detected, five of which were assigned as previously identified 3-methyloxindole, 3-hydroxy-3-methylindolenine, 3-hydroxy-3-methyloxindole, 5-hydroxy-3-methylindole, and 6-hydroxy-3-methylindole. Among the three new metabolites, one was either 4- or 7-OH-3-methylindole, and the other two were derived from additional oxidation on the phenyl ring of 3-methyloxindole. When GSH was added to the microsomal incubations, seven conjugates that had molecular ions corresponding to the incorporation of GSH and an atom of oxygen at m/z 453 (group I) were produced, and two additional conjugates had molecular ions at m/z 437 that corresponded to the incorporation of GSH with no additional oxygen (group II). Two conjugates in group I (m/z 453) were apparently derived by GSH addition to the 5,6-epoxide metabolite of 3-methyloxindole. These two GSH adducts were tentatively identified as 5-(glutathione-S-yl)-3-methyloxindole and 6-(glutathione-S-yl)-3-methyloxindole. The most abundant conjugate in group I was identified as 3-(glutathione-S-yl)-3-methyloxindole, which substantiated the presence of the putative 2,3-epoxy-3-methylindole intermediate. The remaining four adducts in group I were likely formed by conjugation of GSH at different positions of the phenyl ring, possibly via oxidation of 5-hydroxy-3-methylindole and 6-hydroxy-3-methylindole to two very interesting new electrophilic benzoquinone imine intermediates. For the group II conjugates (m/z 437), two isomers were identified as 2-(glutathione-S-yl)-3-methylindole and 3-(glutathione-S-yl-methyl)-indole. The former adduct was primarily derived from the 2,3-epoxide intermediate by thiol conjugation followed by dehydration. The latter adduct was consistent with our previously published work on the dehydrogenation of 3MI. In those studies, we showed that the reactive intermediate, 3-methylenenindolenine, was formed by hydrogen abstraction at the methyl group and was trapped with GSH. The putative dehydrogenation bioactivation mechanism is also substantiated by the finding that CYP2E1 selectively generated 2-(glutathione-S-yl)-3-methylindole but did not produce 3-(glutathione-S-yl-methyl)-indole. In summary, the results not only confirmed the formation of 2,3-epoxide-3-methylindole in human liver microsomes but also suggested that the phenolic metabolites of 3-methylindole were dehydrogenated to previously uncharacterized reactive intermediates.

Substrate analogues for an RNA-editing adenosine deaminase: mechanistic investigation and inhibitor design.

ADARs are adenosine deaminases that act on RNA and are responsible for RNA-editing reactions that occur in eukaryotic mRNAs, including the mRNAs of glutamate and serotonin receptors. ADARs capable of editing biologically relevant RNA substrates have been identified. In addition, the consequence of the RNA-editing reaction on the function of the gene product is known in several cases. However, our understanding of the chemical mechanism of the ADAR-catalyzed adenosine deamination in RNA is lagging. By studying analogues of a naturally occurring substrate for ADAR2, we infer features of the enzyme's active site and reaction mechanism. 8-Aza substitution at adenosine in various RNA substrates accelerates the rate of deamination at these sites by ADAR2 (2.8-17-fold). The magnitude of this "aza effect" depends on the RNA structural context of the reacting nucleotide. N(6)-Methyladenosine in RNA is a slow substrate for ADAR2 (rate is 2% that of adenosine), with no product observed with N(6)-ethyladenosine, suggesting a limited size of the leaving group pocket. 2,6-Diaminopurine ribonucleoside in RNA is not a substrate for ADAR, in contrast to adenosine deaminase (ADA), which catalyzes a similar reaction on nucleosides. This and other results indicate that ADAR2 uses a base recognition strategy different from that of ADA. Consistent with the large 8-aza effect observed for the ADAR2 reaction, we find that 8-azanebularine, as the free nucleoside, inhibits the ADAR2 reaction (IC(50) = 15 +/- 3 mM) with no inhibition observed with nebularine or coformycin.