Inhibition of Insect Cytochromes P450 by Furanocoumarins

Abstract

Coumarin, benzofuran, and 16 furanocoumarins were evaluated as inhibitors of Manduca sexta midgut cytochrome P450-catalyzed O-demethylation of p-nitroanisole. 8- O-Alkylpsoralens with nonpolar alkyl groups of five or fewer carbons had the lowest I 50 values. I 50 values of 5- O-alkylpsoralens increased with the size of the O-alkyl group and were greater than psoralen. Psoralens with polar O-alkyl groups such as alcohols had high I 50 values. Psoralens with substituents on the furan ring were not inhibitory. Coumarin, which lacks a furan ring, was a reversible inhibitor with an I 50 value two orders of magnitude higher than psoralen. Benzofuran which lacks the pyrone ring was a weak mechanism-based inhibitor. The angular furanocoumarin angelicin had an I 50 value comparable to 8- O-alkylpsoralens. All of the inhibitory furanocoumarins tested were mechanism-based irreversible inhibitors. The structure-activity results are consistent with a proposed mechanism of inhibition whereby the furanocoumarin is oxidized by cytochrome P450 at the double bond of the furan ring forming an unstable epoxide that can react with cytochrome P450. [ 3H]Xanthotoxin labels numerous proteins when incubated with microsomes in the presence of NADPH. Although a P450-containing protein fraction separated by chromatography has the highest tritium/protein ratio, the lack of specificity limits the usefulness of radiolabeling with furanocoumarins for identification of cytochromes P450.