Bioactivation of 4-Ipomeanol by CYP4B1:  Adduct Characterization and Evidence for an Enedial Intermediate

Abstract

4-Ipomeanol (IPO) is a pneumotoxin that is bioactivated to a reactive intermediate that binds to DNA and other cellular macromolecules. Despite over 30 years of research in this area, detailed structural information on the nature of the IPO reactive intermediate is still lacking. In the present study, we reacted IPO with rabbit CYP4B1 in the presence of exogenous nucleophiles and analyzed the products by liquid chromatography/electrospray ionization-mass spectrometry. Coincubation of IPO and rabbit CYP4B1 with glutathione gave rise to multiple products due likely to the presence of both sulfur and nitrogen nucleophiles in the same trapping molecule. Reaction mixtures containing equimolar N-acetyl cysteine (NAC) and N-acetyl lysine (NAL) provided a major NADPH- and CYP4B1-dependent product. A combination of high-resolution mass spectrometry and two-dimensional NMR analysis following large-scale isolation of the biologically derived material provided evidence for an N-substituted cysteinyl pyrrole derivative of IPO, analogous to that characterized previously in model chemical studies conducted with cis-2-butene-1,4-dial. Purified native rabbit lung CYP4B1 and purified recombinant rabbit CYP4B1 produced the trapped NAC/NAL-IPO pyrrole adduct at rates of 600-700 nmol/nmol P450/30 min. A panel of 14 commercially available recombinant human CYPs was also studied, and substantial rates of IPO bioactivation (>100 nmol/nmol/30 min) were observed with CYP1A2, CYP2C19, CYP2D6, and CYP3A4. These studies provide evidence for the formation of an enedial reactive intermediate during CYP-mediated IPO bioactivation, identify multiple human liver P450s capable of IPO bioactivation, and demonstrate that the same reactive intermediate is formed by both rabbit CYP4B1 and human P450s.

Ligand characterization of CYP4B1 isoforms modified for high-level expression inEscherichia coliand HepG2 cells

“…Our preliminary identification studies indicate that non-reactive hydroxylated products are the major oxidative metabolites of PK 13, but not of 4-IPO 12 (Supplementary Figure S3). We therefore hypothesize that, in contrast to 4-IPO 12, PK 13 can enter the active site of CYP4B1 in two different binding orientations: the first binding orientation results in non-reactive reaction products through hydroxylation of a carbon atom of the alkyl chain in three possible positions (as confirmed by MS analysis), while the second binding orientation results in (an) reactive enedial intermediate(s) through epoxidation of the furan ring and subsequent rearrangement reactions similar to the bioactivation of 4-IPO 12 (Baer et al, 2005) (as suggested by preliminary results from 'trapping-experiments' with NAC/NAL). However, this alternative pathway does not diminish the cytotoxicity of PK 13 compared to 4-IPO 12 in whole cells, where glucuronidation of 4-IPO 12 is presumably the dominant inactivation route (Parkinson et al, 2016).…”

“…0.71). In separate experiments attempting to trap and identify also the generated reactive cytotoxic metabolites (data not shown), inclusion of NAC and NAL in the metabolic incubations resulted in additional chromatographic peaks with both substrates that were tentatively identified as the NAC/NAL adducts of the cytotoxic metabolites (Supplementary Figure S3) (Baer et al, 2005).…”

“…In wild-type human CYP4B1, this conserved proline should be located at position 427, but is replaced by serine that renders the human enzyme incapable of processing 4-IPO 12 (Baer et al, 2005). Therefore, as humans cannot process 4-IPO 12, Rainov et al (1998) proposed to use the rabbit CYB4B1 as a suicide gene in combination with 4-IPO 12 as pro-drug to treat brain tumors.…”

“…Cheesman et al (2003) were able to modify the N-terminus of the r-P422 enzyme for high-level expression in Escherichia coli and purification of the recombinant protein; in addition, they demonstrated that this recombinant enzyme also has 98% of its heme covalently bound. Using the purified recombinant rabbit enzyme, Baer et al (2005) identified the bioactivation pathway for 4-IPO 12 using N-acetyl cysteine (NAC) and N-acetyl lysine (NAL) as exogenous nucleophiles to trap the formed reactive 4-IPO enedial intermediate in vitro. However, neither the mechanism in vivo nor the endogenous substrate(s) and function(s) of CYP4B1 are known to date.…”

Absorption, Distribution, and Biliary Excretion of Cafestol, a Potent Cholesterol-Elevating Compound in Unfiltered Coffees, in Mice

“…Data from the present study show that the liver is exposed to significant amounts of cafestol, which can lead to the formation of epoxides. This effect has been described for several other furancontaining molecules, including furan, menthofuran, ipomeanine, 4-ipomeanol, furosemide, and teucrin A (Khojasteh-Bakht et al, 1999;Alvarez-Diez and Zheng, 2004;Baer et al, 2005;Peterson et al, 2005;Chen et al, 2006). For some of these compounds, including the natural toxin ipomeanol, formation of furan epoxides has indeed been associated with cellular toxicity.…”