Coumarin Derivatives as Substrate Probes of Mammalian Cytochromes P450 2B4 and 2B6: Assessing the Importance of 7-Alkoxy Chain Length, Halogen Substitution, and Non-Active Site Mutations

Abstract

Using a combined structural and biochemical approach, the functional importance of a recently described peripheral pocket bounded by the E-, F-, G-, and I-helices in CYP2B4 and 2B6 was probed. Three series of 4-substituted-7-alkoxycoumarin derivatives with a -H, -CH3, or -CF3 at the 4 position of the coumarin core were used initially to monitor functional differences between CYP2B4 and 2B6. 7-Ethoxy-4-(trifluoromethyl)coumarin (7-EFC) displayed the highest catalytic efficiency among these substrates. Mutants were made to alter side-chain polarity (V/E194Q) or bulk (F/Y244W) to alter access to the peripheral pocket. Modest increases in catalytic efficiency of 7-EFC O-deethylation by the mutants were magnified considerably by chlorination or bromination of the substrate ethoxy chain. A structure of CYP2B6 Y244W in complex with (+)-α-pinene was solved at 2.2 Å and showed no CYMAL-5 in the peripheral pocket. A ligand free structure of CYP2B4 F244W was solved at 3.0 Å with CYMAL-5 in the peripheral pocket. In both instances, comparison of the respective wild-type and mutant CYP2B enzymes revealed that CYMAL-5 occupancy of the peripheral pocket had little effect on the topology of active site residue side-chains, despite the fact that the peripheral pocket and active site are located on opposite sides of the I-helix. Analysis of available CYP2B structures suggest that the effect of the amino acid substitutions within the peripheral pocket derive from altered interactions between the F and G helices.

So many roads traveled: A career in science and administration

“…Ross Wilderman and Manish Shah moved with me to UConn along with a postdoctoral fellow, Jingbao Liu, a chemist who mastered protein biochemistry and structural biology (49). Manish solved additional structures, including woodrat CYP2B35 and -37 (50), and we thought that his observation of halogen-bonds in numerous CYP2B but not other family 2 enzymes (51) might explain the preference of CYP2B enzymes for halogenated compounds, including the PCBs we had studied in Arizona 30 years before.…”

Coumarins and P450s, Studies Reported to-Date

“…Neither 2A6, nor 2A13, produce 3-hydroxycoumarin during oxidation of coumarin [29]. From the CYP 2B subfamily, only CYP 2B6 is functional in humans [55]. P450 2B6 has been shown to efficiently O-deethylate 7-ethoxy-4-trifluoromethylcomarin [50].…”

“…Crystal structures of P450s 2B4 and 2B6 have been shown to only contain one 4-CPI molecule in their active sites [56,57]. P450s 2B4 and 2B6 also show coumarin substrate selectivity based on the side chain length at position 7, as well as substitution at position 4 [55]. These observations confirm that P450 2B family enzymes show significant differences within their flexible active sites and substrate access channels across various species, and bind a wide range of ligand sizes and shapes [31].…”

Novel Trifluoromethylcoumarinyl Urea Derivatives: Synthesis, Characterization, Fluorescence, and Bioactivity

“…Thus, the introduction of a CF 3 group into bioactive molecules has become a preferable approach in pharmaceutical studies [ 48 , 49 ]. However, the literature about CF 3 -substituted coumarins is limited, and the reports about these compounds are mainly focused on their use as substrate probes to detect the physiology [ 50 , 51 , 52 ]. Also, in view of these observations and in continuation of our research program on the synthesis of fluorinated coumarins [ 53 ], we herein report the synthesis and characterization of some novel 4-trifluoromethyl-6-substituted-ureido-7-methoxylcoumarins, whose fluorescent properties and antifungal and herbicidal activities are also evaluated.…”