Biotransformation, Using Recombinant CYP450-Expressing Baker’s Yeast Cells, Identifies a Novel CYP2D6.10^A122V Variant Which Is a Superior Metabolizer of Codeine to Morphine Than the Wild-Type Enzyme

Abstract

CYP2D6, a cytochrome P450 (CYP) enzyme, metabolizes codeine to morphine. Within the human body, 0–15% of codeine undergoes O-demethylation by CYP2D6 to form morphine, a far stronger analgesic than codeine. Genetic polymorphisms in wild-type CYP2D6 (CYP2D6-wt) are known to cause poor-to-extensive metabolism of codeine and other CYP2D6 substrates. We have established a platform technology that allows stable expression of human CYP genes from chromosomal loci of baker’s yeast cells. Four CYP2D6 alleles, (i) chemically synthesized CYP2D6.1, (ii) chemically synthesized CYP2D6-wt, (iii) chemically synthesized CYP2D6.10, and (iv) a novel CYP2D6.10 variant CYP2D6-C (i.e., CYP2D6.10 A122V ) isolated from a liver cDNA library, were cloned for chromosomal integration in yeast cells. When expressed in yeast, CYP2D6.10 enzyme shows weak activity compared with CYP2D6-wt and CYP2D6.1 which have moderate activity, as reported earlier. Surprisingly, however, the CYP2D6-C enzyme is far more active than CYP2D6.10. More surprisingly, although CYP2D6.10 is a known low metabolizer of codeine, yeast cells expressing CYP2D6-C transform >70% of codeine to morphine, which is more than twice that of cells expressing the extensive metabolizers, CYP2D6.1, and CYP2D6-wt. The latter two enzymes predominantly catalyze formation of codeine’s N-demethylation product, norcodeine, with >55% yield. Molecular modeling studies explain the specificity of CYP2D6-C for O-demethylation, validating observed experimental results. The yeast-based CYP2D6 expression systems, described here, could find generic use in CYP2D6-mediated drug metabolism and also in high-yield chemical reactions that allow the formation of regio-specific dealkylation products.

Cyclohexane-1,3-dione Derivatives as Future Therapeutic Agents for NSCLC: QSAR Modeling, In Silico ADME-Tox Properties, and Structure-Based Drug Designing Approach

“…In terms of metabolism, the cytochrome CYP450 enzymes are responsible for removing toxins from the body . This is done through the oxidation of intrusive foreign substances, including drugs.…”

A Review of the Important Role of CYP2D6 in Pharmacogenomics

“…CYP2D6 has numerous endogenous substrates including tyramine in the brain and lymphocytes [31,32], and 5-hydroxyindoleacetic acid in brain tissue and possibly cerebrospinal fluid [33,34]. Importantly, although CYP2D6 constitutes just 2-4% of total hepatic CYP content [35], it is a cardinal drug-metabolising enzyme involved in the metabolism of approximately 20% of commonly used drugs [36,37]. Thus, CYP2D6 can metabolise a wide range of substrates including analgesics (e.g., codeine, tramadol), antidepressants (e.g., paroxetine, tricyclic antidepressants), antihypertensives (e.g., metoprolol, bisoprolol) and the anti-cancer agent, tamoxifen [38][39][40][41].…”

CYP2D6, Its Genetic Polymorphism, and the Brain

“…Ultrarapid metabolizers of the CYP2D6 have been shown to carry up to 13 copies of the active gene (Bertilsson et al 2002). CYP2D6 accounts for the metabolism of nearly 25% of the clinically used drugs and almost 100 commonly used drugs in the clinics are the substrates for this isozyme despite its low content (2 -4%) among other CYP isozymes in the liver (Bertilsson et al 2002, Chaudhry et al 2014, Williams et al 2018) (Fig. 1 and Fig.…”