Development of new Coumarin-based profluorescent substrates for human cytochrome P450 enzymes

Abstract

1. Cytochrome P450 (CYP) enzymes constitute an essential xenobiotic metabolizing system that regulates the elimination of lipophilic compounds from the body. Convenient and affordable assays for CYP enzymes are important for assessing these metabolic pathways. 2. In this study, 10 novel profluorescent coumarin derivatives with various substitutions at carbons 3, 6 and 7 were developed. Molecular modeling indicated that 3phenylcoumarin offers an excellent scaffold for the development of selective substrate compounds for various human CYP forms, as they could be metabolized to fluorescent 7-hydroxycoumarin derivatives. Oxidation of profluorescent coumarin derivatives to fluorescent metabolites by 13 important human liver xenobiotic-metabolizing CYP forms was determined by enzyme kinetic assays. 3. Four of the coumarin derivatives were converted to fluorescent metabolites by CYP1 family enzymes, with 6-methoxy-3-(4-trifluoromethylphenyl)coumarin being oxidized selectively by CYP1A2 in human liver microsomes. Another set of four compounds were metabolized by CYP2A6 and CYP1 enzymes. 7-Methoxy-3-(3methoxyphenyl)coumarin was oxidized efficiently by CYP2C19 and CYP2D6 in a nonselective fashion. 4. The advantages of the novel substrates were 1) an excellent signal-to-background ratio, 2) selectivity for CYP1 forms, and 3) convenient multiwell plate measurement, allowing for precise determination of potential inhibitors of important human hepatic forms CYP1A2, CYP2C19 and CYP2D6.

Inhibition of human CYP1 enzymes by a classical inhibitor α‐naphthoflavone and a novel inhibitor N‐(3, 5‐dichlorophenyl)cyclopropanecarboxamide: An in vitro and in silico study

“…Synthesis and experimental data for compounds 1 – 6 (3‐(4‐trifluoromethylphenyl)‐6‐methoxycoumarin ( 1 ), 3‐(4‐trifluoromethoxyphenyl)‐7‐methoxycoumarin ( 2 ), 3‐(3‐hydroxyphenyl)‐6‐hydroxycoumarin ( 3) , 3‐(3‐methoxyphenyl)‐7‐methoxycoumarin ( 4) , 3‐(3‐methoxyphenyl)‐6‐methoxycoumarin ( 5) , 3‐(3‐benzyloxyphenyl)‐7‐methoxycoumarin ( 6 ; Figure 1b) have been published earlier (Juvonen, Ahinko, Huuskonen, Raunio, & Pentikainen, 2019; Niinivehmas et al, 2018; Rauhamäki et al, 2018).…”

“…DCPCC was also tested directly for its inhibition potency toward all major human xenobiotic‐metabolizing enzymes. We used the profluorescent 7‐ethoxyresorufin and novel coumarin derivatives as model substrates (Figure 1b; Juvonen, Ahinko, Huuskonen, Raunio, & Pentikäinen, 2018). In silico modeling approaches were used to characterize the binding modes of DCPCC and ANF at the active sites of CYP1A1, CYP1A2, and CYP1B1.…”

Coumarin-Based Profluorescent and Fluorescent Substrates for Determining Xenobiotic-Metabolizing Enzyme Activities In Vitro

“…We recently developed 10 novel profluorescent coumarin derivatives with various substitutions at carbons 3, 6, and 7 [ 41 ]. All derivatives were oxidized to corresponding fluorescent 7-hydroxycoumarin metabolites.…”

“…CYP1 and CYP2A enzymes have overlapping substrate preferences. For example, four novel small 3-phenylcoumarins were metabolized efficiently (high intrinsic clearance) by both CYP2A6 and CYP1 enzymes [ 41 ]. The CYP1 family enzymes and CYP2A6 have an optimal size of the active site for catalysis of 3-phenylcoumarins.…”

Fragment‐ and negative image‐based screening of phosphodiesterase 10A inhibitors

“…Because the shape complementarity between the protein's ligand‐binding cavity and the ligand is a key factor in assuring strong binding, the NIB can outperform traditional virtual screening approaches such as flexible molecular docking on a case‐by‐case basis (Niinivehmas et al., , ; Virtanen & Pentikäinen, ). In addition to the benchmark testing, the NIB methodology has been used successfully in tool compound discovery projects for metabolizing enzymes (Juvonen, Ahinko, Huuskonen, Raunio, & Pentikäinen, ). The NIB benefits especially from the Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) postprocessing, which, in general, has been shown to be an efficient way to recognize correct binding poses of small‐molecule ligands (Ahinko, Niinivehmas, Jokinen, & Pentikäinen, ; Niinivehmas et al., ).…”