Spectroscopic observations of β-eudesmol binding to human cytochrome P450 isoforms 3A4 and 1A2, but not to isoforms 2C9, 2C19, and 2D6

Abstract

1. β-Eudesmol (BEU) is a sesquiterpenoid component of Atractylodes lancea with cytotoxic activity against cholangiocarcinoma. Its lipophilic nature makes BEU a likely substrate of human cytochrome P450 (P450) enzymes.

2. Using ligand-binding difference spectroscopy, the affinities of this compound to recombinant CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 were investigated in Escherichia coli membrane preparations.

3. CYP3A4 showed a type I spectral change, with a binding constant Ks of 77 ± 23 (mean ± SD) μM at 0.5 μM P450 (Ks/[P450] ≈ 155). The reference substrate testosterone (TES) and the inhibitor fluconazole bound to the enzyme with apparent affinities of 86 ± 4 μM (type I) and 21 μM (type II), respectively. BEU was bound by CYP3A4 in a non-cooperative manner (Hill coefficient n ≈ 0.8). CYP1A2 showed reverse type I difference spectra with either BEU or caffeine (CAF). The CYP1A2 affinity for BEU was higher (0.23 mM) than for CAF (0.37 mM) but lower than for phenacetin (0.11 mM, type I). BEU did not bind significantly to CYP2C9, CYP2C19, and CYP2D6.

4. Confirmation of metabolic activity and studies on the involvement of other human P450 isoforms are required. Double-beam spectrometry is needed to validate Ks measurements made with a microplate reader.

Keywords:

• β-Eudesmol
• cytochrome P450
• recombinant enzyme
• CYP3A4
• CYP1A2
• CYP2C9
• CYP2C19
• CYP2D6
• difference spectroscopy

Acknowledgements

CYP expression plasmids were a kind gift from Prof. Elisabeth Gillam, University of Queensland, Australia. Ethan Vindvamara confirmed the identity of the pGro7 plasmid. Amornrat Geadkaew Krenc, Nanthawat Kosa, Pitchanee Wattanasiri and Worapapar Treesuppharat were of invaluable assistance with DNA work, sonication, and ultracentrifugation.

Disclosure statement

The authors declare no conflict of interest.

Additional information

Funding

The study was supported by Thammasat University under the Research Grant 5/2562, and by the Center of Excellence in Pharmacology and Molecular Biology of Malaria and Cholangiocarcinoma. Kesara Na-Bangchang is supported by the National Research Council of Thailand under the Research Team Promotion grant [grant number NRCT 820/2563].