tx9b00330_si_001.pdf (1.82 MB)
Structure–Activity Relationships for CYP4B1 Bioactivation of 4‑Ipomeanol Congeners: Direct Correlation between Cytotoxicity and Trapped Reactive Intermediates
journal contribution
posted on 2019-12-04, 14:08 authored by John P. Kowalski, Matthew G. McDonald, Dale Whittington, Miklos Guttman, Michele Scian, Marco Girhard, Helmut Hanenberg, Constanze Wiek, Allan E. RettieCytochrome P450 4B1 (CYP4B1) has been explored as a candidate
enzyme
in suicide gene systems for its ability to bioactivate the natural
product 4-ipomeanol (IPO) to a reactive species that causes cytotoxicity.
However, metabolic limitations of IPO necessitate discovery of new
“pro-toxicant” substrates for CYP4B1. In the present
study, we examined a series of synthetically facile N-alkyl-3-furancarboxamides for cytotoxicity in HepG2 cells expressing
CYP4B1. This compound series maintains the furan warhead of IPO while
replacing its alcohol group with alkyl chains of varying length (C1–C8).
Compounds with C3–C6 carbon chain lengths showed similar potency
to IPO (LD50 ≈ 5 μM). Short chain analogs
(<3 carbons) and long chain analogs (>6 carbons) exhibited reduced
toxicity, resulting in a parabolic relationship between alkyl chain
length and cytotoxicity. A similar parabolic relationship was observed
between alkyl chain length and reactive intermediate formation upon
trapping of the putative enedial as a stable pyrrole adduct in incubations
with purified recombinant rabbit CYP4B1 and common physiological nucleophiles.
These parabolic relationships reflect the lower affinity of shorter
chain compounds for CYP4B1 and increased ω-hydroxylation of
the longer chain compounds by the enzyme. Furthermore, modest time-dependent
inhibition of CYP4B1 by N-pentyl-3-furancarboxamide
was completely abolished when trapping agents were added, demonstrating
escape of reactive intermediates from the enzyme after bioactivation.
An insulated CYP4B1 active site may explain the rarely observed direct
correlation between adduct formation and cell toxicity reported here.