CYP4F2 and CYP4F3B are cytochrome P450 enzymes (CYPs) that catalyze ω-hydroxylation of fatty acids and arachidonic acid and that metabolize certain drugs (1)
. Enzyme assays for CYP4F2 and CYP4F3B typically include a chromatographic separation step that limits ease-of-use and throughput. Thus, there is a need for simple, multiwell plate-based CYP4F2 and CYP4F3 assays for rapid enzyme analyses and inhibitor screening applications.
Luminogenic CYP assays use prosubstrates for the light-generating reaction of firefly luciferase. CYPs convert the prosubstrates to luciferin, which makes light in a second reaction with a luciferase reaction mix called Luciferin Detection Reagent (LDR) (4)
. The amount of light generated is proportional to the amount of luciferin produced by the CYP, and therefore, to CYP enzyme activity. Multiple CYP enzymes are encoded by families of genes in humans and other organisms (6)
. The CYP enzyme selectivity for a given luminogenic substrate depends on the nature of the derivatization on the luciferin structure.
Here we demonstrated that 2-(6-(4-(methylthio)benzyloxy)benzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid, a luciferin derivative referred to as Luciferin-4F2/3, is converted to luciferin, most prominently by CYP4F3B and has substantial activity with CYP4F2. Luciferin-4F2/3 can be used for luminogenic CYP4F3B and CYP4F2 enzyme assays in a multiwell-plate format.
Materials and Methods
The CYP assays were performed using instructions in the P450-Glo™ Assay Technical Bulletin #TB325 and P450-Glo™ Screening Systems Technical Bulletin #TB340. The CYP enzymes used were recombinant human forms in microsomes from insect cells that coexpressed a human CYP cDNA with P450 reductase or P450 reductase plus cytochrome b5 (Gentest™ Supersomes™, BD Biosciences). A 50mM stock solution of Luciferin-4F2/3 (MW = 416.53) was prepared in acidified dimethylsulfoxide (DMSO with 50mM HCl).* The CYP4F2 or CYP4F3B enzyme assays were performed using 25–50µM Luciferin-4F2/3 substrate (Cat.# P1651) or as indicated in figure legends, 50mM KPO4 (pH 7.4), 20nM CYP enzyme coexpressed with P450 reductase and cytochrome b5 (1pmol/50µl reaction), and 1X NADPH Regeneration System (Cat.# V9510).
Assays were assembled and performed in opaque white 96-well plates (e.g., white polystyrene, 96-well plates [Costar Cat.# 3912]). After incubating reactions for 10 or 20 minutes at 37°C, 50µl of Luciferin Detection Reagent (LDR; Cat.# V8920, V8921) was added to each 50µl CYP reaction to stop the reactions and initiate luminescence. The luminescence was read after 20 minutes using the GloMax® 96 Microplate Luminometer (Cat.# E6501) and reported in relative light units (RLU).
For convenience, you can prepare a 4X concentrated enzyme/buffer/substrate mix (200mM KPO4 [pH 7.4], 200µM Luciferin-4F2/3, 80nM CYP4F2 or CYP4F3B)*, 4X concentrated test compound solution (e.g., for CYP inhibition assays) and 2X concentrated NADPH Regeneration System (7)
. For a 50µl reaction in a 96-well plate, combine 12.5µl of the 4X enzyme mixture with 12.5µl of 4X test compound and initiate the reaction by adding 25µl of the NADPH Regeneration System.
*Note: Luciferin-4F2/3 solubility in KPO4 buffer is improved when the 50mM DMSO stock solution is first diluted to an intermediate concentration of 0.2mM in 100mM Tris-HCl (pH 7.5).
Results and Discussion
The putative luminogenic CYP substrate, Luciferin-4F2/3, was initially screened for activity in the luminescent assay format against 21 recombinant human CYP enzymes (Figure 1). Under the conditions used in Figure 1, we observed prominent activity with CYP4F3B, a relatively smaller activity with CYP4F2, and minor activities with CYP4F3A and CYP4A11.
A time-dependent increase in luminescence reflecting luciferin accumulation was observed at room temperature and 37°C for up to 60 minutes (Figure 2).
The CYP4F3B reaction with Luciferin-4F2/3 showed atypical kinetics with sigmoidicity at the low end of the curve, indicating homotropic cooperativity and substrate inhibition at the high end(9)
The CYP4F3B assay with Luciferin-4F2/3 was tested as a tool for measuring CYP4F3B inhibition (Figure 4). Dose-dependent inhibition by 17-octadecynoic acid, a known CYP4F3B inhibitor (10)
, was observed (IC50 = 18.4µM).
Luciferin-4F2/3, a novel luciferin derivative, is a luminogenic cytochrome P450 substrate with a high degree of selectivity for CYP4F3B and CYP4F2. Using Luciferin-4F2/3 with a CYP enzyme assay approach harnesses the exquisite sensitivity, selectivity and simplicity of bioluminescence. This provides simple, rapid, multiwell plate-based CYP4F2 and CYP4F3B enzyme assays for analysis and screening.