(S)-EFV ((S)-6-chloro-4-(cyclopropylethynyl)-4-(trifluoromethyl)-1,4-dihydro-2H-benzo[d][1,3]oxazin-2-one) and
all EFV metabolites, as well as analogs, were purchased from Toronto
Research Chemicals (Toronto, ON, Canada) and were >95% pure according
to the manufacturer’s certificates of analysis (Figures S2–S11 ). 1H NMR and 19F NMR spectra of these compounds conformed to their structures. l -Glutamate was from MilliporeSigma (St. Louis, MO), and a 2
mM stock solution was prepared in water. Cholesterol was obtained
from Steraloids (Newport, RI), and 24-hydroxy-[25,26,26,26,27,27,27-2H7]-Cholesterol was from Medical Isotopes (Pelham,
NH). Cholesterol was added from a 1 mM stock in 4.5%, w/v, aqueous
2-hydroxypropyl-β-cyclodextrin (HPCD), and deuterated 24-hydroxyCholesterol
was added from a 0.1 mM stock in methanol. Human truncated Δ(2–50)CYP46A1
with a four-histidine tag on the C terminus and rat cytochrome P450
oxidoreductase were expressed in Escherichia coli and purified as described.60 (link),61 (link) The F405A Δ(2–50)CYP46A1
mutant was generated by using an in vitro QuikChange site-directed
mutagenesis kit (Stratagene, San Diego, CA) according to the instructions.
The correct generation of the desired mutation and absence of undesired
mutations were confirmed by nucleotide sequencing of the entire CYP46A1
coding region as well as by the restriction analysis.
all EFV metabolites, as well as analogs, were purchased from Toronto
Research Chemicals (Toronto, ON, Canada) and were >95% pure according
to the manufacturer’s certificates of analysis (
mM stock solution was prepared in water. Cholesterol was obtained
from Steraloids (Newport, RI), and 24-hydroxy-[25,26,26,26,27,27,27-2H7]-Cholesterol was from Medical Isotopes (Pelham,
NH). Cholesterol was added from a 1 mM stock in 4.5%, w/v, aqueous
2-hydroxypropyl-β-cyclodextrin (HPCD), and deuterated 24-hydroxyCholesterol
was added from a 0.1 mM stock in methanol. Human truncated Δ(2–50)CYP46A1
with a four-histidine tag on the C terminus and rat cytochrome P450
oxidoreductase were expressed in Escherichia coli and purified as described.60 (link),61 (link) The F405A Δ(2–50)CYP46A1
mutant was generated by using an in vitro QuikChange site-directed
mutagenesis kit (Stratagene, San Diego, CA) according to the instructions.
The correct generation of the desired mutation and absence of undesired
mutations were confirmed by nucleotide sequencing of the entire CYP46A1
coding region as well as by the restriction analysis.
