Inactivation of CYP3A4 by SCH 66712 and EMTPP
2095
metabolite assays (Supplemental Fig. 3A). In metabolite assays, the major similarities and differences between inactivation of CYP2D6 and
SCH 66712 product formed by CYP3A4 was a C-mono-oxygenation CYP3A4 by SCH66712 will additionally provide insight into enzyme
on the piperazine/heteroaromatic ring end of SCH 66712—the end of function and specificity.
the molecule that would be positioned best for metabolism based on the
Conclusion
not form significant stabilizing interactions that could hold EMTPP in
The inactivation of CYP3A4 and CYP2D6 is of great clinical sig-
a particular orientation for metabolism, possibly explaining the large
nificance because together they are responsible for the metabolism of
variety of mono-oxygenation products formed from EMTPP by
70% of all pharmaceutic drugs currently on the market. Mechanism-
CYP3A4. Furthermore, the binding free energies from docking ex-
based inactivation of P450s is of particular interest because of its
periments were less favorable for EMTPP than for SCH 66712.
irreversible nature and dependence on enzyme catalysis. SCH 66712
There are many MBIs of CYP3A4 reported in the literature, but only
and EMTPP have been previously identified as a MBI of CYP2D6,
a few of CYP2D6, including only two known apoprotein adductors,
and our study shows SCH 66712 and EMTPP to be inactivators of
SCH 66712 and EMTPP. Inactivation of CYP2D6 by SCH 66712 was
CYP3A4 as well.
more potent than inactivation of CYP3A4, with a partition ratio of 3 and
kinact and KI of 0.032 min21 and 0.55 mM, respectively (Nagy et al.,
Acknowledgments
2011). However, inactivation by EMTPP was similar for both CYP2D6
The authors thank Dr. F.P. Guengerich for making this collaboration pos-
and CYP3A4 (partition ratios of 99 and 94 for CYP2D6 and CYP3A4,
respectively; and kinact and KI values of 0.09 min21 and 5.5 mM for
sible, Drs. P. Hollenberg, H. Zhang, and A. Venter, and G. Cavey for helpful
comments, Dr. M.V. Martin for preparing purified recombinant P450 NADPH-
CYP2D6 versus 0.044 min21 and 11.8 mM for CYP3A4). CYP2D6
reductase and cytochrome b5, and Dr. Mark Hail of Novatia for assistance with
inactivation by SCH 66712 and EMTPP also proceeded with 1:1
binding stoichiometries and no protection from inactivation by free
ProMass software.
radical scavengers (Palamanda et al., 2001; Hutzler et al., 2004; Nagy
Authorship Contributions
et al., 2011).
Participated in research design: Bolles, Fujiwara, Nomeir, Furge.
SCH 66712 and EMTPP are structurally related with piperazine
Conducted experiments: Bolles, Fujiwara, Briggs, Furge.
rings, substituted imidazoles, and fluorinated heteroaromatic rings. To
Contributed new reagents or analytic tools: Nomeir.
Performed data analysis: Bolles, Fujiwara, Furge.
our knowledge, there have been no reports of potent MBI of CYP2D6
and CYP3A4 by the same inactivator [though there are conflicting
reports regarding inactivation by tamoxifen (Sridar et al., 2002; Zhao
et al., 2002)]. Part of the lack of dual inactivators of both CYP3A4
and CYP2D6 is the difference in the presence of potential requisite
nucleophile(s) in the active site. For instance, Vandenbrink et al. (2012)
have shown that while raloxifene is a potent inactivator of CYP3A4,
it does not inactivate CYP2D6. They further showed by structural anal-
ysis that CYP2D6 lacks cysteine residues in the quadrant most likely to
interact with raloxifene and other soft-electrophile inactivators. It seems
likely that SCH 66712 and EMTPP are not adducting at a cysteine since
both 2D6 and 3A4 experience inactivation.
We previously proposed that a quinone could be the reactive elec-
trophile in the mechanism of CYP2D6 inactivation by SCH 66712
(Nagy et al., 2011). However, we were unable to isolate a thiol-adduct
in the presence of NAC or glutathione with SCH 66712. In contrast,
we were able to observe an NAC-EMTPP conjugate in reactions with
CYP3A4 as did Hutzler et al. (2004) with CYP2D6. Hutzler et al.
(2004) showed by NMR that the electrophile of EMTPP that reacted
with NAC had formed on the methylene group of the ethyl substituent
of the imidazole ring. However, they were unable to identify the amino
acid target in CYP2D6.
In our molecular models and those of others, it is not clear that a
thiol such as cysteine would be available in CYP2D6 for inactivation
on the distal side (Vandenbrink et al., 2012). It may be that the ethyl
substituent of EMTPP is not the source of the reactive electrophile that
leads to inactivation, as several other metabolites were also formed by
reactions at other sites in EMTPP and in SCH 66712 by both CYP2D6
and CYP3A4. Furthermore, in activity assays in our present study with
CYP3A4, the addition of NAC did not prevent inactivation by EMTPP,
though a NAC-EMTPP conjugate was captured by MS. No conjugates
of SCH 66712 were observed in reactions with CYP3A4 or previous
studies with CYP2D6 (Nagy et al., 2011). Future studies aimed at iden-
tifying the exact structural characteristic(s) of SCH 66712 (and EMTPP)
that plays a role in the covalent binding of SCH66712 to CYP3A4 and
CYP2D6, and the specific location of the covalent adduction to the
P450 enzymes, will provide great insight to drug design. Comparing the
Wrote or contributed to the writing of the manuscript: Bolles, Fujiwara,
Briggs, Nomeir, Furge.
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