A convenient test system for the identification of CYP4V2 inhibitors.

Purpose: Polymorphisms in the gene that codes for the human cytochrome P450 enzyme CYP4V2 are a cause of Bietti crystalline dystrophy (BCD). Therefore, inhibition of CYP4V2 activity may well be a cause of visual disability. However, monitoring the fatty acid hydroxylation reactions catalyzed by this enzyme is tedious and not well suited for inhibitor screening. Methods: We investigated the use of proluciferin compounds as probe substrates for efficient and convenient determination of CYP4V2 activity. Results: Ten proluciferins were tested for conversion by CYP4V2, and eight were found to be substrates of this enzyme. One point inhibitor assays were performed using luciferin 6' 3-furfuryl ether methyl ester (luciferin-3FEME) as the probe substrate and 12 test compounds. As expected, HET0016 had by far the strongest effect, while two other compounds (including osilodrostat) also displayed statistically significant inhibitory potency. The half maximal inhibitory concentration (IC50) for HET0016 was determined to be 179 nM. A recently identified potent inhibitor of human CYP4Z1 was found not to inhibit CYP4V2. To explore the selectivity of this compound between CYP4Z1 and CYP4V2, we developed a homology model of CYP4V2 and conducted docking experiments. Conclusions: We provide the first protocol for a robust and convenient CYP4V2 inhibitor assay that does not depend on fatty acid analysis but can be simply monitored with luminescence. Moreover, we demonstrate additional evidence for the concern that compounds with CYP-inhibitory properties may inhibit CYP4V2 activity and thus, possibly cause visual disability.

HET0016 attenuates the stemness of breast cancer cells through targeting CYP4Z1.

Ours and other previous studies have shown that CYP4Z1 is specifically and highly expressed in breast cancer, and acts as a promoter for the stemness of breast cancer cells. Here, we explored whether targeting CYP4Z1 could attenuate the stemness of breast cancer cells using HET0016, which has been confirmed to be an inhibitor of CYP4Z1 by us and others. Using the transcriptome-sequencing analysis, we found that HET0016 suppressed the expression of cancer stem cell (CSC) markers and stem cell functions. Additionally, HET0016 indeed reduced the stemness of breast cancer cells, as evident by the decrease of stemness marker expression, CD44+ /CD24- subpopulation with stemness, mammary-spheroid formation, and tumor-initiating ability. Moreover, HET0016 suppressed the metastatic capability through in vitro and in vivo experiments. Furthermore, we confirmed that HET0016 suppressed CYP4Z1 activity, and HET0016-induced inhibition on the stemness and metastasis of breast cancer cells was rescued by CYP4Z1 overexpression. Thus, our results demonstrate that HET0016 can attenuate the stemness of breast cancer cells through targeting CYP4Z1.

Discovery of a novel potent cytochrome P450 CYP4Z1 inhibitor.

Human cytochrome P450 enzyme CYP4Z1 represents a promising target for the treatment of a multitude of malignancies including breast cancer. The most active known non-covalent inhibitor (1-benzylimidazole) only shows low micromolar affinity to CYP4Z1. We report a new, highly active inhibitor for CYP4Z1 showing confirmed binding in an enzymatic assay and an IC50 value of 63 ± 19 nM in stably transfected MCF-7 cells overexpressing CYP4Z1. The new inhibitor was identified by a systematically developed virtual screening protocol. Binding was rationalized using a carefully elaborated 3D pharmacophore hypothesis and thoroughly characterized using extensive molecular dynamics simulations and dynamic 3D pharmacophore (dynophore) analyses. This novel inhibitor represents a valuable pharmacological tool to accelerate characterization of the still understudied CYP4Z1 and might pave the way for a new treatment strategy in CYP4Z1-associated malignancies. The presented in silico model for predicting CYP4Z1 interaction provides novel mechanistic insights and revealed that the drug ozagrel interacts with CYP4Z1.

Design and Characterization of the First Selective and Potent Mechanism-Based Inhibitor of Cytochrome P450 4Z1.

Mammary-tissue-restricted cytochrome P450 4Z1 (CYP4Z1) has garnered interest for its potential role in breast cancer progression. CYP4Z1-dependent metabolism of arachidonic acid preferentially generates 14,15-epoxyeicosatrienoic acid (14,15-EET), a metabolite known to influence cellular proliferation, migration, and angiogenesis. In this study, we developed time-dependent inhibitors of CYP4Z1 designed as fatty acid mimetics linked to the bioactivatable pharmacophore, 1-aminobenzotriazole (ABT). The most potent analogue, 8-[(1H-benzotriazol-1-yl)amino]octanoic acid (7), showed a 60-fold lower shifted-half-maximal inhibitory concentration (IC50) for CYP4Z1 compared to ABT, efficient mechanism-based inactivation of the enzyme evidenced by a KI = 2.2 µM and a kinact = 0.15 min-1, and a partition ratio of 14. Furthermore, 7 exhibited low off-target inhibition of other CYP isozymes. Finally, low micromolar concentrations of 7 inhibited 14,15-EET production in T47D breast cancer cells transfected with CYP4Z1. This first-generation, selective mechanism-based inhibitor (MBI) will be a useful molecular tool to probe the biochemical role of CYP4Z1 and its association with breast cancer.

In Vitro Inhibitory Effects of Sesamin on CYP4F2 Activity.

Sesamin is a major lignan in sesame seeds, and a recent meta-analysis of controlled trials indicated that sesamin intake decreases blood pressure. The antihypertensive effect of sesamin has been suggested to be due to sesamin-mediated suppression of 20-hydroxyeicosatetraenoic acid production catalyzed by CYP4F2. However, the detailed mechanism underlying inhibition of CYP4F2 function by sesamin remains unclear. In this study, the effects of sesamin on catalytic activity of CYP4F2 were investigated in vitro. Sesamin inhibited luciferin-4F2/3 O-dealkylase activity of recombinant human CYP4F2 with an IC50 value of 0.381 µM. When preincubated in the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH) for 20 min, sesamin potentiated the inhibition of CYP4F2 activity. Moreover, kinetic analysis of the inactivation revealed that sesamin showed a preincubation time- and concentration-dependent inhibition of CYP4F2 activity yielding a maximal inactivation rate constant (kinact) value of 0.354 min-1 and half-maximal inhibitory concentration (KI) value of 1.12 µM. The inactivation of CYP4F2 by sesamin required NADPH. These results indicated that sesamin is a mechanism-based inactivator of human CYP4F2.

Effects of Ketoconazole, a CYP4F2 Inhibitor, and CYP4F2*3 Genetic Polymorphism on Pharmacokinetics of Vitamin K1.

The objective of this study was to evaluate whether cytochrome P450 (CYP)4F2 is involved in the exposure of vitamin K1 through a drug interaction study with ketoconazole, a CYP4F2 inhibitor, and a pharmacogenetic study with CYP4F2*3. Twenty-one participants with different CYP4F2*3 polymorphisms were enrolled (8 for *1/*1, 7 for *1/*3, and 6 for *3/*3). All participants were treated twice daily for 5 days with 200 mg of ketoconazole or placebo. Finally, a single dose of 10 mg vitamin K1 was administered, plasma levels of vitamin K1 were measured, and its pharmacokinetics was assessed. Ketoconazole elevated the plasma levels of vitamin K1 and increased the average area under the concentration-time curve (AUCinf ) and peak concentration by 41% and 40%, respectively. CYP4F2*3 polymorphism also affected plasma levels of vitamin K1 and its pharmacokinetics in a gene dose-dependent manner. The average AUCinf value was 659.8 ng·h/mL for CYP4F2*1/*1, 878.1 ng·h/mL for CYP4F2*1/*3, and 1125.2 ng·h/mL for CYP4F2*3/*3 (P = .010). This study revealed that ketoconazole and CYP4F2*3 polymorphism substantially increased the exposure of vitamin K1 in humans. These findings provide a plausible explanation for variations in warfarin dose requirements resulting from interindividual variations in vitamin K1 exposure due to CYP4F2-related drug interactions and genetic polymorphisms.

Discovery of rubiarbonone C as a selective inhibitor of cytochrome P450 4F enzymes.

Cytochrome P450 (CYP) enzymes, particularly CYP4A/4F, are the major ω-hydroxylases of arachidonic acid (AA) that can produce 20-hydroxyeicosatetraenoic acid (20-HETE). Although there are dissimilarities in substrate specificity, tissue distribution, and gene regulation between CYP4A and CYP4F, selective CYP4A or 4F inhibitors are currently unavailable. Therefore, this study was designed to develop CYP4F selective inhibitors using a novel inhibitory assay of 20-HETE formation. The assay was established using pooled human kidney microsomes (HKMs) and human recombinant CYP4 enzymes incubated with 1,2,3,4,5-13C AA (13C5 AA) as a substrate to minimize interference by endogenous AA. The intrinsic clearance (Vmax/Km) values were 9.5 µL/min/mg for HKMs and 0.02, 0.9, and 10.1 µL/min/pmol for CYP4A11, CYP4F2, and CYP4F3B, respectively, which suggests a major role for CYP4F in ω-hydroxylation of AA. To validate the assay, we tested well-known pan-CYP4 inhibitor HET0016 along with 50 compounds derived from natural products. Of the screened compounds, rubiarbonone C showed the most potent inhibitory activity. The 50% inhibitory concentrations of rubiarbonone C against CYP4A11, CYP4F2, and 4F3B were > 50, 4.2, and 4.2 µM, respectively. Moreover, epoxyeicosatrienoic acid formation from 13C5 AA was not inhibited by up to 30 µM rubiarbonone C. Meanwhile, in pooled human liver microsomes, CYP1, 2, and 3 family enzymes involved in drug metabolism were not substantially inhibited by rubiarbonone C. Thus, rubiarbonone C is a selective inhibitor of CYP4F and can be used to discriminate among CYP4 family enzymes and evaluate their roles in physiological and pathophysiological conditions.

Expression and Functional Characterization of Breast Cancer-Associated Cytochrome P450 4Z1 in Saccharomyces cerevisiae.

CYP4Z1 is an "orphan" cytochrome P450 (P450) enzyme that has provoked interest because of its hypothesized role in breast cancer through formation of the signaling molecule 20-hydroxyeicosatetraenoic acid (20-HETE). We expressed human CYP4Z1 in Saccharomyces cerevisiae and evaluated its catalytic capabilities toward arachidonic and lauric acids (AA and LA). Specific and sensitive mass spectrometry assays enabled discrimination of the regioselectivity of hydroxylation of these two fatty acids. CYP4Z1 generated 7-, 8-, 9-, 10-, and 11-hydroxy LA, whereas the 12-hydroxy metabolite was not detected. HET0016, the prototypic CYP4 inhibitor, only weakly inhibited laurate metabolite formation (IC50 ∼15 µM). CYP4Z1 preferentially oxidized AA to the 14(S),15(R)-epoxide with high regioselectivity and stereoselectivity, a reaction that was also insensitive to HET0016, but neither 20-HETE nor 20-carboxy-AA were detectable metabolites. Docking of LA and AA into a CYP4Z1 homology model was consistent with this preference for internal fatty acid oxidation. Thus, human CYP4Z1 has an inhibitor profile and product regioselectivity distinct from most other CYP4 enzymes, consistent with CYP4Z1's lack of a covalently linked heme. These data suggest that, if CYP4Z1 modulates breast cancer progression, it does so by a mechanism other than direct production of 20-HETE.

Efficient substrate screening and inhibitor testing of human CYP4Z1 using permeabilized recombinant fission yeast.

We have established a protocol for the preparation of permeabilized fission yeast cells (enzyme bags) that recombinantly express human cytochrome P450 enzymes (CYPs). A direct comparison of CYP3A4 activity gave an eightfold higher space-time yield for enzyme bag-catalyzed biotransformation as compared to whole-cell biotransformation, even though the total number of cells employed was lower by a factor of 150. Biotransformation of the luminogenic substrate Luciferin-H using CYP2C9-containing enzyme bags proceeded efficiently and stably for 24h. CYP4Z1 is of interest because it is strongly overexpressed both in breast cancer cells and in breast cancer metastases; however, current knowledge about its catalytic properties is very limited. Screening of CYP4Z1-containing enzyme bags with 15 luminogenic substrates enabled us to identify two new hydroxylations and eleven ether cleavage reactions that are catalyzed by CYP4Z1. By far the best substrate found in this study was Luciferin benzyl ether (Luciferin-BE). On the basis of the recently published crystal structure of CYP4B1 we created a new homology model of CYP4Z1 and performed molecular docking experiments, which indicate that all active substrates show a highly similar binding geometry compared to the endogenous substrates. The model predicts that Ser113, Ser222, Asn381, and Ser383 are key hydrogen bonding residues. We also identified five new inhibitors of CYP4Z1: miconazole, econazole, aminobenzotriazole, tolazoline, and 1-benzylimidazole respectively, with the last compound being the most potent giving an IC50 value of 180nM in our test system.

Role of 20-Hydroxyeicosatetraenoic Acid (20-HETE) in Androgen-Mediated Cell Viability in Prostate Cancer Cells.

20-Hydroxyeicosatetraenoic acid (20-HETE) is generated intracellularly through the ω-hydroxylation of arachidonic acid by the cytochrome P450 (in humans, CYP4A11 and CYP4F2). 20-HETE induces mitogenic responses in different cancer cells. The aim of this study was to analyze how 20-HETE impacts cell survival, proliferation, and apoptosis in prostate cancer cells. Incubation of the human androgen-sensitive cells (LNCaP) with 1-10 µM HET0016 (a selective inhibitor of 20-HETE synthesis) reduced cell viability by 49*-64%* (*p < 0.05 vs. control). This was explained by a reduction in cell proliferation (vehicle, 46 ± 3%; 1 µM, 23 ± 3%*; 10 µM, 28 ± 3%*) and by an increase in apoptosis (vehicle, 2.1 ± 0%; 1 µM, 16 ± 4%*; 10 µM, 31 ± 3%*). Furthermore, the increase in LNCaP cell viability induced by dihydrotestosterone (DHT, 0.1 nM) was abrogated by 30*-42%* by 1-10 µM HET0016. Incubation with 20-HETE (5-1000 nM) increased LNCaP cell viability up to 50%*, together with a 70%* reduction in apoptosis. PC-3 (androgen-insensitive) cell viability was not affected by either HET0016 or 20-HETE. In LNCaP cells, HET0016 (10 µM) diminished the expression of androgen receptors (AR): messenger RNA (mRNA) (40%*) and protein (50%*). DHT (10 nM) augmented CYP4F2 protein expression (1.9-fold*) and 20-HETE levels (50%*). Oppositely, enzalutamide (AR antagonist) reduced CYP4F2 mRNA and protein expressions by 30 and 25%, respectively. Thus, intracellular availability of 20-HETE is necessary to sustain LNCaP cell viability. 20-HETE may act as a signaling molecule in the pathways involved in LNCaP cell viability upon stimulation of the AR. This effect may be partially attributed to its role on securing normal AR expression levels that in turn contribute to maintain intracellular levels of 20-HETE.

CYP4Z1 - A Human Cytochrome P450 Enzyme that Might Hold the Key to Curing Breast Cancer.

The human cytochrome P450 (CYP) enzyme CYP4Z1 is a fatty acid hydroxylase which among human CYPs is unique for being much stronger expressed in the mammary gland than in all other tissues. Moreover, it is strongly overexpressed in all subtypes of breast cancer, and some overexpression has also been found in other types of malignancies, such as ovarian, lung, and prostate cancers, respectively. Due to its unique expression pattern it is conceivable that this enzymes' activity might be exploited for a new therapeutic approach. However, the main challenge for a CYP4Z1-based prodrug strategy (CBPS) for the treatment of breast cancer (and possibly other CYP4Z1-positive malignancies) is the identification of candidate prodrugs that can be activated by this enzyme. In this mini-review we summarize the current knowledge about the enzymatic properties of the CYP4Z1 enzyme as well as on the expression pattern of the CYP4Z1 gene in both normal and cancer cells. Moreover, we present the first homology model of this enzyme and give an outlook on its potential use in cancer treatment strategies.

Leukotriene B4 Metabolism and p70S6 Kinase 1 Inhibitors: PF-4708671 but Not LY2584702 Inhibits CYP4F3A and the ω-Oxidation of Leukotriene B4 In Vitro and In Cellulo.

LTB4 is an inflammatory lipid mediator mainly biosynthesized by leukocytes. Since its implication in inflammatory diseases is well recognized, many tools to regulate its biosynthesis have been developed and showed promising results in vitro and in vivo, but mixed results in clinical trials. Recently, the mTOR pathway component p70S6 kinase 1 (p70S6K1) has been linked to LTC4 synthase and the biosynthesis of cysteinyl-leukotrienes. In this respect, we investigated if p70S6K1 could also play a role in LTB4 biosynthesis. We thus evaluated the impact of the p70S6K1 inhibitors PF-4708671 and LY2584702 on LTB4 biosynthesis in human neutrophils. At a concentration of 10 µM, both compounds inhibited S6 phosphorylation, although neither one inhibited the thapsigargin-induced LTB4 biosynthesis, as assessed by the sum of LTB4, 20-OH-LTB4, and 20-COOH-LTB4. However, PF-4708671, but not LY2584702, inhibited the ω-oxidation of LTB4 into 20-OH-LTB4 by intact neutrophils and by recombinant CYP4F3A, leading to increased LTB4 levels. This was true for both endogenously biosynthesized and exogenously added LTB4. In contrast to that of 17-octadecynoic acid, the inhibitory effect of PF-4708671 was easily removed by washing the neutrophils, indicating that PF-4708671 was a reversible CYP4F3A inhibitor. At optimal concentration, PF-4708671 increased the half-life of LTB4 in our neutrophil suspensions by 7.5 fold, compared to 5 fold for 17-octadecynoic acid. Finally, Michaelis-Menten and Lineweaver-Burk plots indicate that PF-4708671 is a mixed inhibitor of CYP4F3A. In conclusion, we show that PF-4708671 inhibits CYP4F3A and prevents the ω-oxidation of LTB4 in cellulo, which might result in increased LTB4 levels in vivo.

Food Polyphenol Apigenin Inhibits the Cytochrome P450 Monoxygenase Branch of the Arachidonic Acid Cascade.

The product of cytochrome P450 monooxygenase (P450) ω-hydroxylation of arachidonic acid (AA), 20- hydroxyeicosatetraenoic acid (HETE), is a potent vasoconstrictor. Utilizing microsomes as well as individual CYP4 isoforms we demonstrate here that flavonoids can block 20-HETE formation. Apigenin inhibits CYP4F2 with an IC50 value of 4.6 µM and 20-HETE formation in human liver and kidney microsomes at 2.4-9.8 µM. Interestingly, the structurally similar naringenin shows no relevant effect on the formation of 20-HETE. Based on these in vitro data, it is impossible to evaluate if a relevant blockade of 20-HETE formation can result in humans from intake of polyphenols with the diet. However, the potency of apigenin is comparable to those of P450 inhibitors such as ketoconazole. Moreover, an IC50 value in the micromolar range is also described for the inhibition of CYP-mediated drug metabolism leading to food-drug interactions. The modulation of the arachidonic acid cascade by food polyphenols therefore warrants further investigation.