Ligand characterization of CYP4B1 isoforms modified for high-level expression inEscherichia coliand HepG2 cells

Abstract

Human CYP4B1, a cytochrome P450 monooxygenase predominantly expressed in the lung, inefficiently metabolizes classical CYP4B1 substrates, such as the naturally occurring furan pro-toxin 4-ipomeanol (4-IPO). Highly active animal forms of the enzyme convert 4-IPO to reactive alkylating metabolite(s) that bind(s) to cellular macromolecules. By substitution of 13 amino acids, we restored the enzymatic activity of human CYP4B1 toward 4-IPO and this modified cDNA is potentially valuable as a suicide gene for adoptive T-cell therapies. In order to find novel pro-toxins, we tested numerous furan analogs in in vitro cell culture cytotoxicity assays by expressing the wildtype rabbit and variants of human CYP4B1 in human liver-derived HepG2 cells. To evaluate the CYP4B1 substrate specificities and furan analog catalysis, we optimized the N-terminal sequence of the CYP4B1 variants by modification/truncation and established their heterologous expression in Escherichia coli (yielding 70 and 800 nmol·l −1 of recombinant human and rabbit enzyme, respectively). Finally, spectral binding affinities and oxidative metabolism of the furan analogs by the purified recombinant CYP4B1 variants were analyzed: the naturally occurring perilla ketone was found to be the tightest binder to CYP4B1, but also the analog that was most extensively metabolized by oxidative processes to numerous non-reactive reaction products.

Transcriptome Analysis Reveals Differential Expression of Genes Regulating Hepatic Triglyceride Metabolism in Pekin Ducks During Dietary Threonine Deficiency

“…However, acyl-CoA dehydrogenase, short/branched chain ( ACADSB ), acyl-CoA dehydrogenase family member 11 ( ACAD11 ), and cytochrome P450 ( CYP4B ) were downregulated by the Thr-D diet. These genes are involved in fatty acid β-oxidation and ω-oxidation (Vockley et al, 2000; He et al, 2011; Hsu et al, 2017; Roellecke et al, 2017; Scott, 2017). The Thr-D diet downregulated the expression of carnitine palmitoyltransferase 1B ( CPT1B ) compared with the Pair-F diet.…”

Cytochrome P450 4B1 (CYP4B1) as a target in cancer treatment

“…Finally, 2-HepF and 2-AF resulted in nonspecific toxicities in the cells. 68 These findings suggested significant variations with regard to the substrate specificities among the CYP4B1 enzymes, which has been summarized in Table 3.…”

“…67 Furthermore, perilla ketone (PK), isolated from the oil of Perilla frutescens, shares similar chemical structure with 4-IPO, which is also a well-known pneumotoxin. 68 Thus, Roellecke et al developed PK as an alternative prodrug of 4-IPO to be used in this suicide gene system. 67 The following studies carried out by Roellecke group aimed to identify more new alternative prodrugs to be applied in the suicide gene system (HepG2 cells) expressing four different CYP4B1 enzymes, including the rabbit wild type (r-P422), the human wild type (h-S427), the human activated (h-P427), and human reengineered (CYP4B1Pþ12) proteins.…”

“…67 The following studies carried out by Roellecke group aimed to identify more new alternative prodrugs to be applied in the suicide gene system (HepG2 cells) expressing four different CYP4B1 enzymes, including the rabbit wild type (r-P422), the human wild type (h-S427), the human activated (h-P427), and human reengineered (CYP4B1Pþ12) proteins. 68 Nine furan analogs including 4-IPO, PK, 2-AA, 2-furylpentylketon (2-FPK), 2-pentylfuran (2-PenF), 2-hexylfuran (2-HexF), 2-heptylfuran (2-HepF), 3-methylindole (3-MI), and 2-AF were investigated. Firstly, 4-IPO, PK, and 2-AA showed significant and comparable cytotoxic activities in HepG2 cells expressing r-P422, h-P427, as well as CYP4B1Pþ12.…”

Exploring the Critical Components and Therapeutic Mechanisms of Perilla frutescens L. in the Treatment of Chronic Kidney Disease via Network Pharmacology

“…Use of OB ≥30% and DL ≥0.18 as the screening criteria yielded 14 active components. An analysis of the published literature revealed that although (s)carvone (Alasanea and , caffeic acid (Tyszka-Czochara et al, 2018), rosmarinic acid (Colica et al, 2018), caryophellene (Sharma et al, 2016), perilla aldehyde (Song et al, 2018), and perilla ketone (Roellecke et al, 2017) did not meet the aforementioned OB and DL screening criteria, in vivo and in vitro experiments were validated. Therefore, these components were included to result in 19 main active components (C20 Supraene did not retrieve the predicted target, and so was excluded in further research) (Table 1 and Supplementary Figure S1).…”