Cytochrome P450 1B1 Research Articles

Cytochrome P450 2B6 6 distribution among patients living with HIV in Burkina Faso

The function of cytochrome P450 (CYP) enzymes, one of the major catalysts in drug metabolism, is significantly influenced by genetic polymorphisms leading to substantial inter-individual variability in drug response and/ or adverse reactions activity. The aim of this study was to determine the frequency of the CYP2B6 516G>T allele in patients living with HIV receiving treatment at the Medical Center of Camp General Aboubacar Sangoule Lamizana for whom the treatment was changed after the initiation of treatment because of the appearance of side effects. 155 HIV-positive patients were enrolled. Genomic Deoxyribonucleic acid (DNA) extraction from blood was performed using the GenJET® Genomic DNA according to the manufacturer’s instructions. Genotyping for the cytochrome P450 variant alleles was performed using predesigned primers. The amplification was carried out by Polymerase Chain Reaction (PCR), while the differentiation between the alleles was carried out after digestion with restriction enzymes by electrophoresis. The results obtained showed. Among these patients, 61 changed treatments during their follow-up. The majority of the 61 patients, 32 patients, received an initial treatment regimen based on the AZT (3'-Azido-3'-Deoxythymidine)/3TC (Lamivudine)/ NVP(Nevirapine) combination, followed by 18 patients for the TDF (tenofovir)/3TC (lamivudine)/ EFV(Efavirenz) regimens. Currently, the majority of patients, 48 patients, have benefited from a therapeutic regimen based on the TDF (tenofovir)/3TC (lamivudine) /DTG(Dolutegravir) combination, followed by 11 patients for the TDF (tenofovir)/3TC (lamivudine)/EFV(Efavirenz) regimens. The distribution of the major allele CYP2B6*T was 68%, and the minor CYP2B6*G allele was 32%. Heterozygote (GT genotype) and Homozygous mutants (TT genotype) and Heterozygote were 31% and 52,5%. Studies could be continued to set up cytochrome mutation detection kits to anticipate the onset of side effects and adapt treatments.

Design, Synthesis, and Evaluation of 18F-Labeling CYP1B1 PET Tracer Based on 2-Phenylquinazolin

Cytochrome P450 (CYP)1B1 has been identified to be specifically overexpressed in several solid tumors, thus it’s a potential target for the detection of tumors. Based on the 2-Phenylquinazolin CYP1B1 inhibitors, we designed and synthesized several positron emission computed tomography (PET) imaging probes targeting CYP1B1. Through IC50 determinations, most of these probes exhibited good affinity and selectivity to CYP1B1. Considering their affinity, solubility, and their 18F labeling methods, we chose compound 5c as the best candidate. The 18F radiolabeling of [18F] 5c was easy to handle with good radiolabeling yield and radiochemical purity. In vitro and in vivo stability study indicated that probe [18F]5c has good stability. In cell binding assay, [18F]5c could be specifically taken up by tumor cells, especially HCT-116 cells. Although the tumor-blood (T/B) and tumor-muscle (T/M) values and PET imaging results were unsatisfied, it is still possible to develop PET probes targeting CYP1B1 by structural modification on the basis of 5c in the future.

The involvement of hepatic cytochrome P450s in the cytotoxicity of lapatinib.

Lapatinib, an oral tyrosine kinase inhibitor used as a first-line treatment for HER2-positive breast cancer, has been reported to be associated with hepatotoxicity; however, the underlying mechanisms remain unclear. In this study, we report that lapatinib causes cytotoxicity in multiple types of hepatic cells, including primary human hepatocytes, HepaRG cells, and HepG2 cells. A 24-h treatment with lapatinib induced cell cycle disturbances, apoptosis, and DNA damage, and decreased the protein levels of topoisomerase in HepG2 cells. We investigated the role of cytochrome P450 (CYP)-mediated metabolism in lapatinib-induced cytotoxicity using our previously established HepG2 cell lines, which express each of 14 CYPs (1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7). We demonstrate that lapatinib is metabolized by CYP1A1, 3A4, 3A5, and 3A7. Among these, lapatinib-induced cytotoxicity and DNA damage were attenuated in cells overexpressing CYP3A5 or 3A7. Additionally, we measured the production of three primary metabolites of lapatinib (O-dealkylated lapatinib, N-dealkylated lapatinib, and N-hydroxy lapatinib) in CYP1A1-, 3A4-, 3A5-, and 3A7-overexpressing HepG2 cells. We compared the cytotoxicity of lapatinib and its 3 metabolites in primary human hepatocytes, HepaRG cells, and HepG2 cells and demonstrated that N-dealkylated lapatinib is more toxic than the parent drug and the other metabolites. Taken together, our results indicate that lapatinib-induced cytotoxicity involves multiple mechanisms, such as apoptosis and DNA damage; that N-dealkylated lapatinib is a toxic metabolite contributing to the toxic effect of lapatinib; and that CYP3A5- and 3A7-mediated metabolism plays a role in attenuating the cytotoxicity of lapatinib.

Activation of the Wnt/β-catenin/CYP1B1 pathway alleviates oxidative stress and protects the blood-brain barrier under cerebral ischemia/reperfusion conditions.

Abstract JOURNAL/nrgr/04.03/01300535-202407000-00033/figure1/v/2023-11-20T171125Z/r/image-tiff Accumulating evidence suggests that oxidative stress and the Wnt/β-catenin pathway participate in stroke-induced disruption of the blood-brain barrier. However, the potential links between them following ischemic stroke remain largely unknown. The present study found that cerebral ischemia leads to oxidative stress and repression of the Wnt/β-catenin pathway. Meanwhile, Wnt/β-catenin pathway activation by the pharmacological inhibitor, TWS119, relieved oxidative stress, increased the levels of cytochrome P450 1B1 (CYP1B1) and tight junction-associated proteins (zonula occludens-1 [ZO-1], occludin and claudin-5), as well as brain microvascular density in cerebral ischemia rats. Moreover, rat brain microvascular endothelial cells that underwent oxygen glucose deprivation/reoxygenation displayed intense oxidative stress, suppression of the Wnt/β-catenin pathway, aggravated cell apoptosis, downregulated CYP1B1 and tight junction protein levels, and inhibited cell proliferation and migration. Overexpression of β-catenin or knockdown of β-catenin and CYP1B1 genes in rat brain microvascular endothelial cells at least partly ameliorated or exacerbated these effects, respectively. In addition, small interfering RNA-mediated β-catenin silencing decreased CYP1B1 expression, whereas CYP1B1 knockdown did not change the levels of glycogen synthase kinase 3β, Wnt-3a, and β-catenin proteins in rat brain microvascular endothelial cells after oxygen glucose deprivation/reoxygenation. Thus, the data suggest that CYP1B1 can be regulated by Wnt/β-catenin signaling, and activation of the Wnt/β-catenin/CYP1B1 pathway contributes to alleviation of oxidative stress, increased tight junction levels, and protection of the blood-brain barrier against ischemia/hypoxia-induced injury.

Lower overall survival in male patients with advanced disease undergoing allogeneic hematopoietic stem cell transplantation is associated with CYP1B1 Leu432Val polymorphism.

Human cytochrome P450 1B1 (CYP1B1) is an extrahepatic key enzyme involved in estrogen metabolism, steroid synthesis, and pro-carcinogen activation. In a single-center retrospective study, 382 patients who underwent allogeneic hematopoetic stem cell transplantation and their donors were genotyped for CYP1B1 C432G polymorphism by reverse transcription polymerase chain reaction. One hundred and sixty-nine patients (44%) were homozygous wild-type (wt) gene CC, 157 (41%) heterozygous CG and 56 (15%) homozygous gene mutated GG. Of interest, mutated CYP1B1 was more common in male (62%) than in female patients (48%) P=0.006, unlike in donors. Five-year estimate for overall survival (OS) was 58±4% (CC) versus 48±3% (CG and GG), P=0.048. Surprisingly, this difference was only evident in males (P=0.024): OS 58±6% versus 42±4%, whereas it was virtually absent in females. Importantly, this difference was only evident in male patients with advanced disease (AD) (n=118, P=0.002): OS 44±8% (CC) versus 32±6% (CG) versus 6±6% (GG), whereas it was virtually absent in male patients with early disease. One-year non-relapse mortality in male patients with AD was 8±4% (CC) versus 21±5% (CG) versus 50±12% (GG), P=0.002. Three-year relapse rate in male patients with AD was 31±7% (wt) versus 42±6% (mut), P=0.04. Multivariate analysis for OS in male patients with AD revealed CYP1B1 polymorphism as the only prognostic factor: RR 1.78, P=0.001. In conclusion, these results suggest that male patients with AD and mutant CYP1B1 polymorphism have lower OS after allogeneic hematopoetic stem cell transplantation due to a higher non-relapse mortality and a higher relapse rate.

Acetylcholinesterase activity, histopathological changes, lipid peroxidation and stress-related genes expression in Nile tilapia (Oreochromis niloticus) exposed to waterborne methidathion

The safety of aquatic system has been threatened due to extensive and inappropriate application of organophosphorus pesticide methidathion (MD). Hence, a 12-day stress trial was carried out to explore the impacts of waterborne MD on acetylcholinesterase (AChE) activity, histopathology, antioxidant status and stress-related genes expression in Nile tilapia (Oreochromis niloticus). Fish (initial mean weight 60 ± 2 g) were placed in 18 tanks (volume 460 L, 30 fish/tank), with triplicate treatment groups being exposed to diverse concentrations of MD (0, 0.8, 1.67, 5.34, 7.49 and 15.83 µg/L). The results showed that AChE activities in serum and liver were inhibited by MD. After the 12-day stress trial, malonaldehyde (MDA) content in serum and liver was higher than that of 0 µg/L group when MD concentration exceeded 0.8 and 5.34 µg/L, respectively. Catalase (CAT) and total superoxide dismutase (T-SOD) activity in serum and liver revealed irregular variations. In addition, MD (＞0.8 µg/L) caused lesions in gills and liver tissues. MD also down-regulated expression of cytochrome P450 1B1, heat shock protein 70 and cytochrome c oxidase subunit 7B, which contradicted the trend of phospholipase A2 and cytochrome P450 1 A (except in the 15.83 µg/L group). In a word, waterborne MD suppressed AChE activities, induced oxidative stress and harmed the liver and gills. Additionally, it may negatively influence resistance to stress, oxidative phosphorylation, detoxification and anti-inflammatory response in Nile tilapia.

Allelic variants of CYP2B6 gene expression and its implication on the pathogenesis of malaria among a cohort of outpatients in North-Central Nigeria

Abstract Background Human cytochrome P450 2B6 (CYP2B6) is fortified with the biotransformation of the antimalarial, artemisinin combination therapy (ACT). Owing to emerging reports of Plasmodium species resistance to ACT in other climes, CYP2B6*6 genotype significantly alters ACT metabolism. In North-Central Nigeria, the distribution of the CYP2B6*6 variant is poorly documented. This study investigated the distribution of CYP2B6 c.516G>T variants and its relationship with certain malaria pathogenesis among a cohort of clinical-malaria outpatients in Ilorin, Nigeria. Methods A total of 50 symptomatic P. falciparum malaria-positive samples were genotyped for CYP2B6 c.516G>T using restriction fragment length polymorphism and a specific haplotype population was established. The allele frequencies and genotype distributions were analyzed. Haplotypes were clustered using Ward’s method. Correlations determined include defective CYP2B6 versus parasitemia densities and thrombocytopenia, respectively. Results Forty-five samples show genotypic ratios and nine CYP2B6 genetic single nucleotide polymorphisms were identified. The following haplotypes (64C>T = *1/*2, 785A>G = *1/*4, and 1459C>T = *1/*5) occurred and *2, *3, and *18 alleles harbor 64C>T, 777C>A, −82T>C, and 499C>G. The codon 64CT, 516GT, and 785AG; −82TC and 777CA; and 499CG, 516GT, and 785AG were identified as *2/*6 heterozygotes. Samples with 516GT and 785GG genotypes occurred with *4/*6 heterozygotes. Mutant trait alleles recorded high parasitemia 72 h post-ACT regimen. Only individuals with CYP2B6*6 alleles had severe malaria and thrombocytopenia. Conclusion This report contributes to the growing knowledge of CYP2B6*6 genotype frequency and its relationship with malaria pathogenesis among a Nigerian population.

METTL3 exacerbates insulin resistance in hepatocytes by regulating m6A modification of cytochrome P450 2B6

BackgroundInsulin resistance (IR) in hepatocytes endangers human health, and frequently results in the development of non-alcoholic fatty liver disease (NAFLD). Research on m6A methylation of RNA molecules has gained popularity in recent years; however, the molecular mechanisms regulating the processes of m6A modification and IR are not known. The cytochrome P450 (CYP450) enzyme system, which is mainly found in the liver, is associated with the pathogenesis of NAFLD. However, few studies have been conducted on CYP450 related m6A methylation. Here, we investigated the role of the methyltransferase METTL3 in exacerbating IR in hepatocytes, mainly focusing on the regulation of m6A modifications in CYP2B6.Methods and resultsAnalysis using dot blot and epitranscriptomic chips revealed that the m6A modification pattern of the transcriptome in high-fat diet (HFD)-induced fatty liver and free fatty acid (FFA)-induced fatty hepatocytes showed significant changes. CYP450 family members, especially Cyp2b10, whose homolog in humans is CYP2B6, led to a noticeable increase in m6A levels in HFD-induced mice livers. Application of the METTL3 methyltransferase inhibitor, STM2457, increased the level of insulin sensitivity in hepatocytes. We then analyzed the role of METTL3 in regulating m6A modification of CYP2B6 in hepatocytes. METTL3 regulated the m6A modification of CYP2B6, and a positive correlation was found between the levels of CYP2B6 translation and m6A modifications. Furthermore, interference with METTL3 expression and exposure to STM2457 inhibited METTL3 activity, which in turn interfered with the phosphorylated insulin receptor substrate (pIRS)-glucose transporter 2 (GLUT2) insulin signaling pathway; overexpression of CYP2B6 hindered IRS phosphorylation and translocation of GLUT2 to membranes, which ultimately exacerbated IR.ConclusionThese findings offer unique insights into the role that METTL3-mediated m6A modifications of CYP2B6 play in regulating insulin sensitivity in hepatocytes and provide key information for the development of strategies to induce m6A modifications for the clinical treatment of NAFLD.

2,2',4,4'-Tetrabromodiphenyl ether and cadmium co-exposure activates aryl hydrocarbon receptor pathway to induce ROS and GSDME-dependent pyroptosis in renal tubular epithelial cells.

We have previously found that a mixture exposure of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) and cadmium (Cd) causes kidney damage; however, the mechanism was not fully understood. The aryl hydrocarbon receptor (AhR) is a ligand-receptor transcription factor that plays an important role in the adaptive response or metabolic detoxification of environmental toxins. Thus, this study aimed to examine the role of AhR in kidney toxicity. BDE-47 (50 μM) or Cd (5 μM) exposure reduced cell viability in renal tubular epithelial cells (HKC), with a larger effect observed in co-treatment. The cell morphology presented pyroptotic changes, including swollen cells, large bubbles, and plasma membrane pore formation. The gene expressions of AhR, heat shock protein 90 (Hsp90), AhR nuclear translocator (ARNT), and cytochrome P450 1B1 (CYP1B1) were increased, while CYP1A1 was decreased. Reactive oxygen species (ROS) were generated, which was reduced by the AhR antagonist CH223191. The apoptosis, necrosis, and intracellular lactated hydrogenase (LDH) release was elevated, and this was attenuated by N-acetylcysteine (NAC). Furthermore, the pyroptosis pathway was activated with increased protein levels of cleaved-caspase-3 and gasdermin E N-terminal (GSDME-NT), while caspase-8, caspase-3, and GSDME were decreased. These effects were alleviated by NAC and CH223191. Our data demonstrate a combined effect of BDE-47 and Cd on nephrotoxicity by activating AhR to induce ROS contributing to GSDME-dependent pyroptosis, and retardation of the AhR pathway could reduce this toxicity.

Enhancing the invasive traits of breast cancers by CYP1B1 via regulation of p53 to promote uPAR expression

Human cytochrome P450 1B1 (CYP1B1) catalyzes estrogen metabolism to produce metabolites that promote the progression of breast cancer. Since the invasive properties of cancer cells cause cancer relapse, which dramatically reduces patient survival, we investigated the new pro-invasive mechanism involving CYP1B1 in breast cancer. Exploring clinical data from invasive breast cancer patients revealed that CYP1B1 exhibits a potential correlation with urokinase-type plasminogen activator receptor (uPAR). Interestingly, uPAR mRNA expression was elevated in invasive breast cancer patients carrying TP53 genes with driver mutations, and our results showed that CYP1B1 activates the uPAR pathway following regulation of p53 according to its mutant status. CYP1B1 suppressed wild-type (WT) p53 whereas it induced the oncogenic gain-of-function mutant p53R280K, not only via transcriptional regulation but also the protein stabilization and activation following phosphorylation on Ser15 residue of p53R280K. Intriguingly, results from CYP1B1 polymorphic gene study and 4-hydroxyestradiol (4-OHE2) treatment showed that CYP1B1 regulates p53s and uPAR through its enzymatic activity. Furthermore, effects of DMBA and TMS on uPAR expression disappeared in HCT116p53−/− cells, indicating that p53 is critical for uPAR induction by CYP1B1. Collectively, our results demonstrate that CYP1B1 may reduce the relapse-free survival rate of breast cancer patients by inducing invasive traits in cancer cells via p53 regulation based on the mutation status of TP53 genes and further activation of the uPAR pathway. The elucidation of the previously unknown molecular mechanism of CYP1B1 may provide evidence for the development of effective anti-cancer therapeutic strategies that target the progression of cancer invasion.

Abstract P384: Cytochrome P450 1B1-Testosterone Metabolite 6β-Hydroxytestosterone Promotes Ang II-induced Hypertension And Neuroinflammation Via Group IV Cytosolic Phospholipase A 2 α In Paraventricular Nucleus Of Male Mice

We reported previously that Ang (angiotensin) II-induced hypertension depends on the activation of group IV cPLA 2 α (cytosolic phospholipase A 2 α). Moreover, we showed that testosterone (T) metabolite 6β-hydroxytestosterone (6β-OHT) generated by CYP1B1 (cytochrome P450 1B1) in the paraventricular nucleus (PVN) promotes Ang II-induced hypertension and neuroinflammation in male mice. This study determined the relationship of cPLA 2 α and CYP1B1-T derived 6β-OHT in the PVN to Ang II-induced hypertension and neuroinflammation in male mice. cPLA 2 α knockdown in the PVN by transduction with adenovirus (Ad)-cPLA 2 α-short hairpin (sh)RNA (200 nL, bilaterally, 5.4X10 11 pfu/mL) but not its control Ad-scrambled (Scr)-shRNA (2.5X10 11 pfu/mL) in wild-type (WT) male mice, inhibited Ang II (700 ng/kg/min, s.c., two weeks)-induced increase in systolic blood pressure (Day 12- SBP mmHg, tail-cuff) (126±4 vs 167±4, respectively, P<0.05, n=4/group). Moreover, reconstitution of cPLA 2 α in the PVN by Ad-cPLA 2 α-DNA (5.5X10 9 pfu/mL), but not its control Ad-GFP-DNA (1.0X10 11 pfu/mL) in cPLA 2 α -/- mice restored the increase in SBP (153±6 vs 117±4, respectively, P<0.05, n=4/group). Ang II-induced increase in mean arterial blood pressure (MAP mmHg, radiotelemetry) that was minimized in WT-orchidectomized (ORX) or intact Cyp1b1 -/- mice were restored by ICV injections of 6β-OHT (1.5 μg/2 μL/alternate day), but it was attenuated by ICV (2 μL, single injection) Ad-cPLA 2 α-shRNA, but not its control Ad-Scr-shRNA (114±4 vs 150±7, and 121±2 vs 154±9, respectively, P<0.05, n=4-6/group). Ang II-induced increase in MAP was restored by 6β-OHT, but not its vehicle (2-hydroxypropyl-β-cyclodextrin in CSF, 2 μL/alternate day) in Ad-cPLA 2 α-DNA transduced ORX- cPLA 2 α -/- mice (165±6 vs 121±2, respectively, P<0.05, n=4/group). The number of TMEM119 + microglia and GFAP + astrocytes in PVN, markers of neuroinflammation, followed the changes in BP in the above groups produced by Ang II. In conclusion, T-CYP1B1 metabolite 6β-OHT promotes Ang II-induced hypertension and neuroinflammation by a mechanism dependent on cPLA 2 α in the PVN. Therefore, agents that reduce CYP1B1 or cPLA 2 α activity could be useful for treating Ang II/T-dependent hypertension and neuroinflammation in males.

Multi-omics approaches revealed the therapeutic mechanisms of Suo-Quan-Wan for treating overactive bladder in spontaneously hypertensive rats

Ethnopharmacological relevanceSuo-Quan-Wan (SQW), a traditional Chinese prescription, has been used for hundreds of years to alleviate overactive bladder (OAB) symptoms such as frequent and nocturnal urination. However, limited modern research on OAB therapeutic targets has hindered the use and development of SQW. Aim of the studyThis study aimed to investigate the biological mechanisms and key targets of SQW on OAB in spontaneously hypertensive rats (SHR) using an integrated analysis of network pharmacology, transcriptome and metabolome. MethodsRats were divided into five groups: model group (SHR), control group (WKY), darifenacin group, high dose (SQWH) and low dose (SQWL) group. Urodynamic parameters and histological examination were detected. Network pharmacology, transcriptome, and metabolome were used to screen for disease gene targets, differential mRNA, and differential metabolites, respectively. The biological targets and mechanisms of SQW for OAB were analyzed. Western blotting was performed to verify the proteins of key differential targets. ResultsUrodynamics revealed a significant decrease in storage parameters in SHR. After SQW treatment, the inter-contraction interval, voided volume and bladder capacity increased by 2–3 times, as well as bladder compliance. Additionally, SQW improved the pathological changes in the urinary tract epithelium and the detrusor layer of the bladder in SHR. Metabolomic results showed an increase in arachidonic acid (AA) and cyclic adenosine monophosphate (cAMP) in plasma, suggesting the involvement of arachidonic acid metabolism and purine metabolism in SQW treatment. The downregulation of cytochrome P450 1B1 (CYP1B1), thromboxane-A synthase (TBXAS1), polyunsaturated fatty acid 5-lipoxygenase (ALOX5), and cAMP-specific 3′,5′-cyclic phosphodiesterase 4B (PDE4B) were confirmed through topological analysis and Venn analysis of omics data and network pharmacology. These proteins affected the metabolism of AA and cAMP, respectively, and consequently affected downstream proteins, such as transient receptor potential (TRP) cation channel proteins (e.g. TRPV1, TRPA1, and TRPM8), myosin light chain kinase (MLCK), and the phosphorylation of myosin regulatory light chain (p-MLC). ConclusionThis study initially elucidated the importance of AA and cAMP in the treatment of SQW, indicating the AA-CYP1B1/TBXAS1/ALOX5-TRPA1/TRPV1/TRPM8 and cAMP-PDE4B-MLCK-p-MLC pathways as the important pathways in SQW-treated SHR bladder in vivo.

Hepatic biotransformation of non-psychotropic phytocannabinoids and activity screening on cytochromes P450 and UDP-glucuronosyltransferases

This study examined the biotransformation of phytocannabinoids in human hepatocytes. The susceptibility of the tested compounds to transformations in hepatocytes exhibited the following hierarchy: cannabinol (CBN) > cannabigerol (CBG) > cannabichromene (CBC) > cannabidiol (CBD). Biotransformation included hydroxylation, oxidation to a carboxylic acid, dehydrogenation, hydrogenation, dehydration, loss/shortening of alkyl, glucuronidation and sulfation. CBN was primarily metabolized by oxidation of a methyl to a carboxylic acid group, while CBD, CBG and CBC were preferentially metabolized by direct glucuronidation. The study also screened for the activity of recombinant human cytochromes P450 (CYPs) and UDP-glucuronosyltransferases (UGTs), which could catalyze the hydroxylation and glucuronidation of the tested compounds, respectively. We found that CBD was hydroxylated mainly by CYPs 2C8, 2C19, 2D6; CBN by 1A2, 2C9, 2C19 and 2D6; and CBG by 2B6, 2C9, 2C19 and 2D6. CBC exhibited higher susceptibility to CYP-mediated transformation than the other tested compounds, mainly with CYPs 1A2, 2B6, 2C8, 2C19, 2D6 and 3A4 being involved. Further, CBD was primarily glucuronidated by UGTs 1A3, 1A7, 1A8, 1A9 and 2B7; CBN by 1A7, 1A8, 1A9 and 2B7; CBG by 1A3, 1A7, 1A8, 1A9, 2B4, 2B7 and 2B17; and the glucuronidation of CBC was catalyzed by UGTs 1A1, 1A8, 1A9 and 2B7.

Direct interaction between the transmembrane helices stabilize cytochrome P450 2B4 and cytochrome b5 redox complex

The catalytic activity of cytochrome P450 2B4 (CYP2B4) is moderated by its cognate redox partner cytochrome b5 (Cyt-b5). The endoplasmic reticulum (ER) membrane and intermolecular transmembrane (TM) interaction between CYP2B4 and Cyt-b5 regulate the substrate catalysis and the reaction rate. This emphasizes the significance of elucidating the molecular basis of CYP2B4 and Cyt-b5 complexation in a membrane environment to better understand the enzymatic activity of CYP2B4. Our previous solid-state NMR studies revealed the membrane topology of the transmembrane domains of these proteins in the free and complex forms. Here, we show the cross-angle complex formation by the single-pass TM domains of CYP2B4 and Cyt-b5, which is mainly driven by several salt-bridges (E2-R128, R21-D104 and K25-D104), using a multi-microsecond molecular dynamic simulation. Additionally, the leucine-zipper residues (L8, L12, L15, L18 and L19 from CYP2B4) and π-stacking between H23 and F20 residues of CYP2B4 and W110 of Cyt-b5 are identified to stabilize the TM-TM complex in the ER membrane. The simulated tilts of the helices in the free and in the complex are in excellent agreement with solid-state NMR results. The TM-TM packing influences a higher order structural stability when compared to the complex formed by the truncated soluble domains of these two proteins. MM/PBSA based binding free energy estimates nearly 100-fold higher binding affinity (ΔG = -2810.68 ± 696.44 kJ/mol) between the soluble domains of the full-length CYP2B4 and Cyt-b5 when embedded in lipid membrane as compared to the TM-domain-truncated soluble domains (ΔG = -27.406 ± 10.32 kJ/mol). The high-resolution full-length CYP2B4-Cyt-b5 complex structure and its dynamics in a native ER membrane environment reported here could aid in the development of approaches to effectively modulate the drug-metabolism activity of CYP2B4.

Association between CYP2B6 genetic variability and cyclophosphamide therapy in pediatric patients with neuroblastoma

Cyclophosphamide, an oxazaphosphorine prodrug is frequently used in treatment of neuroblastoma, which is one of the most prevalent solid organ malignancies in infants and young children. Cytochrome P450 2B6 (CYP2B6) is the major catalyst and CYP2C19 is the minor enzyme in bioactivation and inactivation pathways of cyclophosphamide. CYP-mediated metabolism may contribute to the variable pharmacokinetics of cyclophosphamide and its toxic byproducts leading to insufficient response to the therapy and development of clinically significant side effects. The aim of the study was to reveal the contribution of pharmacogenetic variability in CYP2B6 and CYP2C19 to the treatment efficacy and cyclophosphamide-induced side effects in pediatric neuroblastoma patients under cyclophosphamide therapy (N = 50). Cyclophosphamide-induced hematologic toxicities were pivotal in all patients, whereas only moderate hepatorenal toxicity was developed. The patients’ CYP2B6 metabolizer phenotypes were associated with the occurrence of lymphopenia, thrombocytopenia, and monocytopenia as well as of liver injury, but not with kidney or urinary bladder (hemorrhagic cystitis) toxicities. Furthermore, the patients’ age (< 1.5 years, P = 0.03) and female gender (P ≤ 0.02), but not CYP2B6 or CYP2C19 metabolizer phenotypes appeared as significant prognostic factors in treatment outcomes. Our results may contribute to a better understanding of the impact of CYP2B6 variability on cyclophosphamide-induced side effects.

16R-HETE and 16S-HETE alter human cytochrome P450 1B1 enzyme activity probably through an allosteric mechanism.

Cytochrome P450 1B1 (CYP1B1) has been widely associated with the development of cardiac pathologies due to its ability to produce cardiotoxic metabolites like midchain hydroxyeicosatetraenoic acids (HETEs) from arachidonic acid (AA) through an allylic oxidation reaction. 16-HETE is a subterminal HETE that is also produced by CYP-mediated AA metabolism. 19-HETE is another subterminal HETE that was found to inhibit CYP1B1 activity, lower midchain HETEs, and have cardioprotective effects. However, the effect of 16-HETE enantiomers on CYP1B1 has not yet been investigated. We hypothesized that 16(R/S)-HETE could alter the activity of CYP1B1 and other CYP enzymes. Therefore, this study was carried out toinvestigate the modulatory effect of 16-HETE enantiomers on CYP1B1 enzyme activity, and to examine the mechanisms by which they exert these modulatory effects. To investigate whether these effects are specific to CYP1B1, we also investigated 16-HETE modulatory effects on CYP1A2. Our results showed that 16-HETE enantiomers significantly increased CYP1B1 activity in RL-14 cells, recombinant human CYP1B1, and human liver microsomes, as seen by the significant increase in 7-ethoxyresorufin deethylation rate. On the contrary, 16-HETE enantiomers significantly inhibited CYP1A2 catalytic activity mediated by the recombinant human CYP1A2 and human liver microsomes. 16R-HETE showed stronger effects than 16S-HETE. The sigmoidal binding mode of the enzyme kinetics data demonstrated that CYP1B1 activation and CYP1A2 inhibition occurred through allosteric regulation. In conclusion, our study provides the first evidence that 16R-HETE and 16S-HETE increase CYP1B1 catalytic activity through an allosteric mechanism.

Machine Learning Models to Predict Cytochrome P450 2B6 Inhibitors and Substrates.

Cytochrome P450 2B6 (CYP2B6) is responsible for the metabolism of ∼7% of marketed drugs. The in vitro drug interaction studies guidance for industry issued by the FDA stipulates that drug sponsors need to evaluate whether the investigated drugs interact with the major drug-metabolizing P450s including CYP2B6. Therefore, there has been greater attention to the development of predictive models for CYP2B6 inhibitors and substrates. In this study, conventional machine learning and deep learning models were developed to predict CYP2B6 inhibitors and substrates. Our results showed that the best CYP2B6 inhibitor model yielded the AUC values of 0.95 and 0.75 with the 10-fold cross-validation and the test set, respectively, and the best CYP2B6 substrate model produced the AUC values of 0.93 and 0.90 with the 10-fold cross-validation and the test set, respectively. The generalization ability of the CYP2B6 inhibitor and substrate models was assessed by using the external validation sets. Several significant substructural fragments relevant to CYP2B6 inhibitors and substrates were detected via frequency substructure analysis and information gain. In addition, the applicability domain of the models was defined by employing a nonparametric method based on the probability density distribution. We anticipate that our results would be useful for the prediction of potential CYP2B6 inhibitors and substrates in the early stage of drug discovery.

Mitochondrial cytochrome P450 1B1 is involved in pregnenolone synthesis in human brain cells

Neurosteroids, which are steroids synthesized by the nervous system, can exert neuromodulatory and neuroprotective effects via genomic and nongenomic pathways. The neurosteroid and major steroid precursor pregnenolone has therapeutical potential in various diseases, such as psychiatric and pain disorders, and may play important roles in myelination, neuroinflammation, neurotransmission, and neuroplasticity. Although pregnenolone is synthesized by CYP11A1 in peripheral steroidogenic organs, our recent study showed that pregnenolone must be synthesized by another mitochondrial cytochrome P450 (CYP450) enzyme other than CYP11A1 in human glial cells. Therefore, we sought to identify the CYP450 responsible for pregnenolone production in the human brain. Upon screening for CYP450s expressed in the human brain that have mitochondrial localization, we identified three enzyme candidates: CYP27A1, CYP1A1, and CYP1B1. We found that inhibition of CYP27A1 through inhibitors and siRNA knockdown did not negatively affect pregnenolone synthesis in human glial cells. Meanwhile, treatment of human glial cells with CYP1A1/CYP1B1 inhibitors significantly reduced pregnenolone production in the presence of 22(R)-hydroxycholesterol. We performed siRNA knockdown of CYP1A1 or CYP1B1 in human glial cells and found that only CYP1B1 knockdown significantly decreased pregnenolone production. Furthermore, overexpression of mitochondria-targeted CYP1B1 significantly increased pregnenolone production under basal conditions and in the presence of hydroxycholesterols and low-density lipoprotein. Inhibition of CYP1A1 and/or CYP1B1 via inhibitors or siRNA knockdown did not significantly reduce pregnenolone synthesis in human adrenal cortical cells, implying that CYP1B1 is not a major pregnenolone-producing enzyme in the periphery. These data suggest that mitochondrial CYP1B1 is involved in pregnenolone synthesis in human glial cells.

Loss of cytochrome P450 (CYP)1B1 mitigates hyperoxia response in adult mouse lung by reprogramming metabolism and translation

Oxygen supplementation is life saving for premature infants and for COVID-19 patients but can induce long-term pulmonary injury by triggering inflammation, with xenobiotic-metabolizing CYP enzymes playing a critical role. Murine studies showed that CYP1B1 enhances, while CYP1A1 and CYP1A2 protect from, hyperoxic lung injury. In this study we tested the hypothesis that Cyp1b1-null mice would revert hyperoxia-induced transcriptomic changes observed in WT mice at the transcript and pathway level. Wild type (WT) C57BL/6J and Cyp1b1-null mice aged 8–10 weeks were maintained in room air (21% O2) or exposed to hyperoxia (>95% O2) for 48h. Transcriptomic profiling was conducted using the Illumina microarray platform. Hyperoxia exposure led to robust changes in gene expression and in the same direction in WT, Cyp1a1-, Cyp1a2-, and Cyp1b1-null mice, but to different extents for each mouse genotype. At the transcriptome level, all Cyp1-null murine models reversed hyperoxia effects. Gene Set Enrichment Analysis identified 118 hyperoxia-affected pathways mitigated only in Cyp1b1-null mice, including lipid, glutamate, and amino acid metabolism. Cell cycle genes Cdkn1a and Ccnd1 were induced by hyperoxia in both WT and Cyp1b1-null mice but mitigated in Cyp1b1-null O2 compared to WT O2 mice. Hyperoxia gene signatures associated positively with bronchopulmonary dysplasia (BPD), which occurs in premature infants (with supplemental oxygen being one of the risk factors), but only in the Cyp1b1-null mice did the gene profile after hyperoxia exposure show a partial rescue of BPD-associated transcriptome. Our study suggests that CYP1B1 plays a pro-oxidant role in hyperoxia-induced lung injury.

Discovery of 4’-trifluoromethylchalcones as novel, potent and selective hCYP1B1 inhibitors without concomitant AhR activation

Human cytochrome P450 1B1 (hCYP1B1), an extrahepatic cytochrome P450 enzyme over-expressed in various tumors, has been validated as a promising target for preventing and treating cancers. Herein, two series of chalcone derivatives were synthesized to discover potent hCYP1B1 inhibitors without AhR agonist effect. Structure-activity relationship (SAR) studies demonstrated that 4′-trifluoromethyl on the B-ring strongly enhanced the anti-hCYP1B1 effects, identifying A9 as a promising lead compound. Further SAR analysis on A9 derivatives (modified A-ring of 4′-trifluoromethylchalcone) showed that introducing 2-methoxyl improved the anti-hCYP1B1 effect and selectivity, while introducing a methoxyl at the C-4 site was beneficial for avoiding AhR activation. Ultimately, five 4′-trifluoromethyl chalcones were identified as potent hCYP1B1 inhibitors (IC50 < 10 nM), while B18 exhibits the most potent anti-hCYP1B1 effect (IC50 = 3.6 nM), suitable metabolic stability and good cell-permeability. B18 also acted as an AhR antagonist and could down-regulate hCYP1B1 in living systems. Mechanistic studies showed that B18 potently inhibited hCYP1B1 in a competitive inhibition manner (Ki = 3.92 nM), while docking simulations revealed that B18 could tightly bind to the catalytic cavity of hCYP1B1 mainly via hydrophobic and hydrogen-bonding interactions. Furthermore, B18 could potently inhibit hCYP1B1 in living cells and showed remarkable anti-migration ability on MFC-7 cells. Taken together, this study deciphered the SARs of chalcones as hCYP1B1 inhibitors and provided several potent hCYP1B1 inhibitors as promising candidates for the development of more efficacious anti-migration agents.