A rapid screening for cytochrome P450 catalysis on new chemical entities: cytochrome P450 BM3 and 1,2,5-oxadiazole derivatives.

This work presents the validation of a rapid screening procedure for the catalysis of cytochrome P450 on new chemical entities. The assay is tested on the prototypical, catalytically self-sufficient and soluble cytochrome P450 BM3 from Bacillus megaterium that shares a high degree of homology with mammalian counterparts. The so-called alkali assay developed in our laboratory is validated here also by product formation and molecular modeling on a number of derivatives sharing the molecular scaffold of the 1,2,5-oxadiazole ring, a class of molecules very different from the long-chain fatty acids known to be oxidized by cytochrome P450 BM3. The alkali assay reveals the ability of this cytochrome to oxidize NADPH in the presence of nine out of thirteen 1,2,5-oxadiazole derivatives tested. The enzyme shows high affinity and coupling efficiencies when incubated with four 1,2,5-oxadiazole derivatives. The presence of oxidation products deriving from catalysis was also confirmed by high-performance liquid chromatography (HPLC). Molecular docking suggests that a key factor for the 1,2,5-oxadiazole derivatives to enter the active site and induce catalysis is the presence of the -SO(2) moiety bridging the 1,2,5-oxadiazole and phenyl rings. These data indicate that the alkali assay is able to quickly and cheaply detect the recognition of new substrates by cytochrome P450. The assay is not intended to substitute HPLC-mass spectrometry analysis, but it is a preliminary screening that allows elimination of obvious nonsubstrates from the start.