Substrate Selectivity of Coumarin Derivatives by Human CYP1 Enzymes: In Vitro Enzyme Kinetics and In Silico Modeling

Abstract

Of the three enzymes in the human cytochrome P450 family 1, CYP1A2 is an important enzyme mediating metabolism of xenobiotics including drugs in the liver, while CYP1A1 and CYP1B1 are expressed in extrahepatic tissues. Currently used CYP substrates, such as 7-ethoxycoumarin and 7-ethoxyresorufin, are oxidized by all individual CYP1 forms. The main aim of this study was to find profluorescent coumarin substrates that are more selective for the individual CYP1 forms. Eleven 3-phenylcoumarin derivatives were synthetized, their enzyme kinetic parameters were determined, and their interactions in the active sites of CYP1 enzymes were analyzed by docking and molecular dynamic simulations. All coumarin derivatives and 7-ethoxyresorufin and 7-pentoxyresorufin were oxidized by at least one CYP1 enzyme. 3-(3-Methoxyphenyl)-6-methoxycoumarin ( 19 ) was 7-O-demethylated by similar high efficiency [21–30 ML/(min·mol CYP)] by all CYP1 forms and displayed similar binding in the enzyme active sites. 3-(3-Fluoro-4-acetoxyphenyl)coumarin ( 14 ) was selectively 7-O-demethylated by CYP1A1, but with low efficiency [0.16 ML/(min mol)]. This was explained by better orientation and stronger H-bond interactions in the active site of CYP1A1 than that of CYP1A2 and CYP1B1. 3-(4-Acetoxyphenyl)-6-chlorocoumarin ( 20 ) was 7-O-demethylated most efficiently by CYP1B1 [53 ML/(min·mol CYP)], followed by CYP1A1 [16 ML/(min·mol CYP)] and CYP1A2 [0.6 ML/(min·mol CYP)]. Variations in stabilities of complexes between 20 and the individual CYP enzymes explained these differences. Compounds 14 , 19 , and 20 are candidates to replace traditional substrates in measuring activity of human CYP1 enzymes.

Coumarins as Tool Compounds to Aid the Discovery of Selective Function Modulators of Steroid Hormone Binding Proteins

“…However, for the best three HSD1 inhibitors, 2 , 10 , 11 , only the most potent HSD1 inhibitor 11 blocks CYP1A2 at an alarming level [ 8 ]. 3-phenylcoumarins have been widely studied as profluorescent substrates for variety of CYP enzymes: compounds 2 , 8 , 11 , 18 – 19 , 29 (compounds 7 , 12 , 4 , 8 – 10 ; respectively in [ 33 ]) and 4 , 14 , 16 , 20 (compounds 15 , 23 , 13 , 21 ; respectively in [ 34 ]). A wide panel of studied CYP enzymes and 3-phenylcoumarin compounds show that these compounds have the potential to act as tool molecules when the metabolism of small molecules is under investigation.…”

Hydration and Structural Adaptations of the Human CYP1A1, CYP1A2, and CYP1B1 Active Sites by Molecular Dynamics Simulations

“…On the other hand, a mobile network of water molecules in the binding pocket can also destabilize the ligand binding. Among the CYP1 isozymes, more water molecules were found in CYP1A1 than in CYP1A2 and CYP1B1, according to the binding site volume [19]. The least number of water molecules occurred in CYP1A2, and this isoform was the most efficient in 7-hydroxylation of 3-phenylcoumarin derivatives; in this case, an excess of water molecules did not destabilize the substrate binding [19].…”

“…Water molecules are supposed to play an important role in the interaction of ligands in the binding sites of enzymes; however, in most studies, the presence of water molecules in the binding cavity is omitted. Recently, in the study of interactions of 3-phenylcoumarin derivatives with CYP1 isoforms, the hydrogen bonds via water molecules were shown with MD simulations [19]. On the other hand, a mobile network of water molecules in the binding pocket can also destabilize the ligand binding.…”

“…Molecular docking and molecular dynamics simulations allow us to explain the functions of enzymes, the role of access channels, and the flexibility of both the binding site of the enzyme and the more distant parts of the protein, which influence the substrate specificity. Interactions of substrates in the binding sites of CYP1 family members were explored with the use of several ligands: series of coumarin derivatives [19], trans-stilbene derivatives [20][21][22][23][24], flavonoids and α-naphthoflavone derivatives [25][26][27][28], and a series of eight compounds-mostly therapeutic agents differing in the specificity toward CYP1s [29]. The selective activity of inhibitors toward CYP1 isoforms is currently being studied with the use of molecular dynamics simulations.…”

“…Molecular dynamics simulations allow the movements of water molecules to be followed and reciprocal interactions between a ligand, water molecules, and a target protein. There are only a few reports on water molecules' role in the ligand binding to CYP1 isoforms [19,35,36]. Failure to include water molecules in CYP1 docking is a potential source of error in estimating ligand affinity by scoring functions.…”