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Cytochrome P450 Enzymes for the Synthesis of Novel and Known Drugs and Drug Metabolites
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2019
Sanjana Haque, Yuqing Gong, Sunitha Kodidela, Mohammad A. Rahman, Sabina Ranjit, Santosh Kumar
According to recent FDA guidelines, if >10% of a parent drug forms a metabolite, that metabolite will need to undergo separate toxicity testing. For this purpose, large quantities of the drug metabolite is needed (FDA, 2016). Therefore, recent studies highlighted the broad applications of engineered CYPBM3 in the production of human metabolites (Girvan and Munro, 2016). For example, the Munro group engineered CYPBM3 by rational mutagenesis to facilitate the binding of the proton pump inhibitor, omeprazole. The mutations of F87V and A82F resulted in a conformational alternation and a decreased free energy barrier, ultimately changing the substrate recognition. These mutants of CYPBM3 were able to transform omeprazole to the human CYP2C19-type metabolite (Butler et al., 2013). The same group further demonstrated the efficient transformation of several proton pump inhibitors (e.g., lansoprazole, esomeprazole) to human CYP-type metabolites by CYPBM3 F87V/A82F mutants (Butler et al., 2014). In a recent work, a library of CYPBM3 variants carrying multiple mutations that were identified from directed evolutions showed a high oxidation activity for a wide range of drugs. Few of these drugs include, non-steroidal anti-inflammatory drugs/NSAIDs (diclofenac and naproxen), muscle relaxant (chlorzoxazone), antidepressant (amitriptyline), anesthetic, antiarrhythmic drug (lidocaine), and steroid hormones (testosterone). Importantly, CYPBM3 mutants from this library converted diclofenac with 91–100% conversion, and produced human CYP2C9-type and CYP3A4-type metabolites, 4’-hydroxylated and 5’-hydroxylated diclofenac with 34% yield and 47% yield, respectively (Ren et al., 2015). In another recent example, CYPBM3 mutant M11, has been proven to produce human metabolites of fenamic acid NSAIDs including mefenamic acid, meclofenamic acid, and tolfenamic acid (Venkataraman et al., 2014; Capoferri et al., 2016). Mutant M11 was generated by using random mutagenesis and had 90-fold higher initial activities compared with human CYP2D6 (van Vugt-Lussenburg et al., 2007). This mutant was able to synthesize benzylic or aromatic hydroxylation metabolites of the fenamic acid NSAIDs with high substrate conversions and high turnover numbers (2000–6000). Because of the high total turnover numbers, M11 can be used as a biocatalytic tool for large-scale production of fenamic acid metabolites to perform characterization study, activity study, and toxicological evaluation of NSAIDs (Venkataraman et al., 2014). Similarly, mutant M11 was capable of metabolizing anticancer drugs cyclophosphamide and ifosfamide, producing active metabolites. It can also be used for extracellular bioactivation, and for providing a catalytically efficient alternative to liver S9 fraction for toxicity evaluation (Vredenburg et al., 2015). In addition, a set of BM3 mutants, generated using both rational and random mutagenesis, was able to produce human metabolites of 17β-estradiol. This engineered CYPBM3 enzyme can catalyze same reactions as human CYP1A1, CYP1A2, and CYP1B1 with high catalytic efficiency (kcat/Km) and total turnover numbers between 1040 and 1210, producing human metabolites for industrial applications (Cha et al., 2014).
Review on age-specific exposure to organophosphate esters: Multiple exposure pathways and microenvironments
Published in Critical Reviews in Environmental Science and Technology, 2023
Jia-Yong Lao, Yuefei Ruan, Kenneth M. Y. Leung, Eddy Y. Zeng, Paul K. S. Lam
OPEs can be biodegraded in human body by phase I and phase II metabolisms, and some of these metabolites are excreted via urine (Hou et al., 2016). As metabolites, DnBP, BCEP, BCIPP, BDCIPP, DPhP, and BBOEP are commonly paired with TnBP, TCEP, TCIPP, TDCIPP, TPhP, and TBOEP, respectively, for body burden estimation (Chen et al., 2018; Fromme et al., 2014; Hou et al., 2016; Li, Dong, et al., 2019). The molar fraction of metabolite to parent OPE (Fue) is a crucial parameter for this estimation, but has undergone limited investigation. Van den Eede et al. (2013) estimated the Fue values of BCEP, BCIPP, BDCIPP, DPhP, and BBOEP to be 0.07, 0.33, 0.46, 0.22, and 0.81, respectively, by human liver microsome (HLM) and 0.13, 0.28, 0.68, 0.2, and 0.16, respectively, by human liver S9 fraction. Wang et al. (2020) calculated the average Fue values of DnBP, BCEP, BCIPP, BDCIPP, DPhP, and BBOEP which were 0.05, 0.42, 0.27, 0.69, 0.42, and 0.03, respectively, by HLM. These results indicate that TDCIPP has a higher clearance rate through urinary BDCIPP excretion. The inconsistent values of Fue of individual OPEs point to the need for accurate estimation of body burden.
The expression of Phase II drug-metabolizing enzymes in human B-lymphoblastoid TK6 cells
Published in Journal of Environmental Science and Health, Part C, 2022
Xilin Li, Yuxi Li, Kylie G. Ning, Si Chen, Lei Guo, Jessica A. Bonzo, Nan Mei
In a previous study, we demonstrated that TK6 cells without any exogenous enzyme system were able to methylate luteolin into diosmetin. Diosmetin showed lower genotoxicity and cytotoxicity compared to its parental form luteolin.11 After a 24-h exposure, more than 90% of luteolin in the medium was converted to diosmetin measured by liquid chromatography with tandem mass spectrometry (LC-MS/MS). This observation made us question whether some traditionally recognized “metabolically incompetent” cell lines may possess exogenous drug/chemical metabolizing enzymes. As shown in Table 1 and Figure 1A, TK6 cells had a basal level expression of COMT at both the mRNA and protein levels, further confirming our postulation in the previous study that COMT present in TK6 cells accounted for the biotransformation of luteolin. HepG2 cells and PHHs also showed high expression of COMT in both mRNA and protein levels, which is consistent with previous findings.13,30 The implication of COMT in genotoxicity assessment is mainly associated with its O-methylation reaction in flavonoids. As seen in the case of quercetin, one of the most well-studied flavonoids, COMT is likely to explain the in vitro and in vivo discrepancy in its carcinogenic effects. Quercetin is highly mutagenic in vitro, but not in animals. COMT was proposed to rapidly methylate quercetin and provides sufficient inactivation in vivo.31 Since such a detoxification process is missing or less efficient in the Ames test, quercetin constantly produces positive results with or without the rat liver S9 fraction. Mammalian cell models such as TK6 cells and HepG2 cells constitutively express COMT, and thus may serve as complementary in vitro tools to the Ames test for studying the genotoxicity of flavonoids.