P450s 3A4 Research Articles

Design, Synthesis, and Biological Studies of Flavone-Based Esters and Acids as Potential P450 2A6 Inhibitors.

As a potential means for smoking cessation and consequently prevention of smoking-related diseases and mortality, in this study, our goal was to investigate the inhibition of nicotine metabolism by P450 2A6. Smoking is the main cause of many diseases and disabilities and harms nearly every organ of the body. As reported by the Centers for Disease Control and Prevention (CDC), more than 16 million Americans are living with diseases caused by smoking. On average, the life expectancy of a smoker is about 10 years less than a nonsmoker. Smoking cessation can substantially reduce the incidence of smoking-related diseases, including cancer. At least, 70 of the more than 7000 cigarette smoke components, including polycyclic aromatic hydrocarbons, N-nitrosamines, and aromatic amines, are known carcinogens. Nicotine is the compound responsible for the addictive and psychopharmacological effects of tobacco. Cytochrome P450 enzymes are responsible for the phase I metabolism of many tobacco components, including nicotine. Nicotine is mainly metabolized by cytochrome P450s 2A6 and 2A13 to cotinine. This metabolism decreases the amount of available nicotine in the bloodstream, leading to increased smoking behavior and thus exposure to tobacco toxicants and carcinogens. Here, we report the syntheses and P450 2A6 inhibitory activities of a number of new flavone-based esters and acids. Three of the flavone derivatives studied were found to be potent competitive inhibitors of the enzyme. Docking studies were used to determine the possible mechanisms of the activity of these inhibitors.

Cytochrome P450 Enzymes as Drug Targets in Human Disease.

Although the mention of cytochrome P450 (P450) inhibition usually brings to mind unwanted variability in pharmacokinetics, in several cases P450s are good targets for inhibition. These P450s are essential, but in certain disease states, it is desirable to reduce the concentrations of their products. Most of the attention to date has been with human P450s 5A1, 11A1, 11B1, 11B2, 17A1, 19A1, and 51A1. In some of those cases, there are multiple drugs in use, e.g., exemestane, letrozole, and anastrozole with P450 19A1, the steroid aromatase target in breast cancer. There are also several targets that are less developed, e.g., P450s 2A6, 8B1, 4A11, 24A1, 26A1, and 26B1. SIGNIFICANCE STATEMENT: The selective inhibition of certain cytochrome P450s that have major physiological functions has been shown to be very efficacious in certain human diseases. In several cases, the search for better drugs continues.

Exercise alleviates diabetic complications by inhibiting oxidative stress-mediated signaling cascade and mitochondrial metabolic stress in GK diabetic rat tissues.

Type 2 diabetes, obesity (referred to as "diabesity"), and metabolic syndrome associated with increased insulin resistance and/or decreased insulin sensitivity have been implicated with increased oxidative stress and inflammation, mitochondrial dysfunction, and alterations in energy metabolism. The precise molecular mechanisms of these complications, however, remain to be clarified. Owing to the limitations and off-target side effects of antidiabetic drugs, exercise-induced control of hyperglycemia and increased insulin sensitivity is a preferred strategy to manage "diabesity" associated complications. In this study, we have investigated the effects of moderate exercise (1h/day, 5days a week for 60days) on mitochondrial, metabolic, and oxidative stress-related changes in the liver and kidney of type 2 diabetic Goto-Kakizaki (GK) rats. Our previous study, using the same exercise regimen, demonstrated improved energy metabolism and mitochondrial function in the pancreas of GK diabetic rats. Our current study demonstrates exercise-induced inhibition of ROS production and NADPH oxidase enzyme activity, as well as lipid peroxidation and protein carbonylation in the liver and kidney of GK rats. Interestingly, glutathione (GSH) content and GSH-peroxidase and GSH reductase enzymes as well as superoxide dismutase (SOD) activities were profoundly altered in diabetic rat tissues. Exercise helped in restoring the altered GSH metabolism and antioxidant homeostasis. An increase in cytosolic glycolytic enzyme, hexokinase, and a decrease in mitochondrial Kreb's cycle enzyme was observed in GK diabetic rat tissues. Exercise helped restore the altered energy metabolism. A significant decrease in the activities of mitochondrial complexes and ATP content was also observed in the GK rats and exercise regulated the activities of the respiratory complexes and improved energy utilization. Activation of cytochrome P450s, CYP 2E1, and CYP 3A4 was observed in the tissues of GK rats, which recovered after exercise. Altered expression of redox-responsive proteins and translocation of transcription factor NFκB-p65, accompanied by activation of AMP-activated protein kinase (AMPK), SIRT-1, Glut-4, and PPAR-γ suggests the induction of antioxidant defense responses and increased energy metabolism in GK diabetic rats after exercise.

Evaluation of MTBH, a novel hesperetin derivative, on the activity of hepatic cytochrome P450 isoform in vitro and in vivo using a cocktail method by HPLC-MS/MS

ABSTRACT 8-methylene-tert-butylamine-3',5,7-trihydroxy-4'-methoxyflavanone (MTBH), a novel hesperidin derivative, has potential in the prevention of hepatic disease, however, its effects on cytochrome P450 isoforms (P450s) remains unexplored. The purpose was to investigate the effects of MTBH on the mRNA, protein levels and activities of six P450s (1A2, 2C11/9, 2D2/6, 3A1/4, 2C13/19 and 2E1) in vitro and in vivo. In vitro study, rat and human liver microsomes were adopted to elucidate the inhibitory effect of MTBH on six P450s using probe drugs. In vivo study, Sprague-Dawley male rats were treated with MTBH (25, 50 or 100 mg/kg for 28 consecutive days), phenobarbital (80 mg/kg for 12 consecutive days) or 0.5% CMC-Na solution (control group) by intragastric administration, then, the mRNA, protein levels and activities of liver P450s were analyzed by real-time PCR, western blotting and probe-drug incubation systems, respectively. The in vitro study indicated that MTBH inhibits the activities of CYP3A1/4 and CYP2E1 in rat and human liver microsomes. In vivo data showed that MTBH inhibits mRNA, protein levels and activities of CYP3A1 and CYP2E1 in medium- and high-dose MTBH groups. MTBH has the potential to cause drug-drug interactions when co-administered with drugs that are metabolized by CYP3A1/4 and CYP2E1.

Cytochrome b 5 Binds Tightly to Several Human Cytochrome P450 Enzymes.

Numerous studies have been reported in the past 50-plus years regarding the stimulatory role of cytochrome b 5 (b 5) in some, but not all, microsomal cytochrome P450 (P450) reactions with drugs and steroids. A missing element in most of these studies has been a sensitive and accurate measure of binding affinities of b 5 with P450s. In the course of work with P450 17A1, we developed a fluorescent derivative of a human b 5 site-directed mutant, Alexa 488-T70C-b 5, that could be used in binding assays at sub-μM concentrations. Alexa 488-T70C-b 5 bound to human P450s 1A2, 2B6, 2C8, 2C9, 2E1, 2S1, 4A11, 3A4, and 17A1, with estimated K d values ranging from 2.5 to 61 nM. Only weak binding was detected with P450 2D6, and no fluorescence attenuation was observed with P450 2A6. All of the P450s that bound b 5 have some reported activity stimulation except for P450 2S1. The affinity of P450 3A4 for b 5 was decreased somewhat by the presence of a substrate or inhibitor. The fluorescence of a P450 3A4•Alexa 488-T70C-b 5 complex was partially restored by titration with NADPH-P450 reductase (POR) (K d,apparent 89 nM), suggesting the existence of a ternary P450 3A4-b 5-POR complex, as observed previously with P450 17A1. Gel filtration evidence was also obtained for this ternary complex with P450 3A4. Overall, the results indicated that the affinity of b 5 for many P450s is very high, and that ternary P450-b 5-POR complexes are relevant in P450 3A4 reactions as opposed to a shuttle mechanism. SIGNIFICANCE STATEMENT: High-affinity binding of cytochrome b 5 (b 5) (K d < 100 nM) was observed with many drug-metabolizing cytochrome P450 (P450) enzymes. There is some correlation of binding with reported stimulation, with several exceptions. Evidence is provided for a ternary P450 3A4-b 5-NADPH-P450 reductase complex.

Differential Reversible and Irreversible Interactions between Benzbromarone and Human Cytochrome P450s 3A4 and 3A5.

Mounting evidence has revealed that despite the high degree of sequence homology between cytochrome P450 3A isoforms (i.e., CYP3A4 and CYP3A5), they have the propensities to exhibit vastly different irreversible and reversible interactions with a single substrate. We have previously established that benzbromarone (BBR), a potent uricosuric agent used in the management of gout, irreversibly inhibits CYP3A4 via mechanism-based inactivation (MBI). However, it remains unelucidated if CYP3A5-its highly homologous counterpart-is susceptible to inactivation by BBR. Using three structurally distinct probe substrates, we consistently demonstrated that MBI was not elicited in CYP3A5 by BBR. Our in silico covalent docking models and molecular dynamics simulations suggested that disparities in the susceptibilities toward MBI could be attributed to the specific effects of BBR covalent adducts on the F-F' loop. Serendipitously, we also discovered that BBR reversibly activated CYP3A5-mediated rivaroxaban hydroxylation wherein apparent V max increased and K m decreased with increasing BBR concentration. Fitting data to the two-site model yielded interaction factors α and β of 0.44 and 5.88, respectively, thereby confirming heterotropic activation of CYP3A5 by BBR. Furthermore, heteroactivation was suppressed by the CYP3A inhibitor ketoconazole in a concentration-dependent manner and decreased with increasing preincubation time, implying that activation was incited via binding of parent BBR molecule within the enzymatic active site. Finally, noncovalent docking revealed that CYP3A5 can more favorably accommodate both BBR and rivaroxaban in concert as compared with CYP3A4, which further substantiated our experimental observations. SIGNIFICANCE STATEMENT: Although it has been previously demonstrated that benzbromarone (BBR) inactivates CYP3A4, it remains uninterrogated whether it also elicits mechanism-based inactivation in CYP3A5, which shares ∼85% sequence similarity with CYP3A4. This study reported that BBR exhibited differential irreversible and reversible interactions with both CYP3A isoforms and further unraveled the molecular determinants underpinning their diverging interactions. These data offer important insight into differential kinetic behavior of CYP3A4 and CYP3A5, which potentially contributes to interindividual variabilities in drug disposition.

Human Family 1-4 cytochrome P450 enzymes involved in the metabolic activation of xenobiotic and physiological chemicals: an update.

This is an overview of the metabolic activation of drugs, natural products, physiological compounds, and general chemicals by the catalytic activity of cytochrome P450 enzymes belonging to Families 1-4. The data were collected from > 5152 references. The total number of data entries of reactions catalyzed by P450s Families 1-4 was 7696 of which 1121 (~ 15%) were defined as bioactivation reactions of different degrees. The data were divided into groups of General Chemicals, Drugs, Natural Products, and Physiological Compounds, presented in tabular form. The metabolism and bioactivation of selected examples of each group are discussed. In most of the cases, the metabolites are directly toxic chemicals reacting with cell macromolecules, but in some cases the metabolites formed are not direct toxicants but participate as substrates in succeeding metabolic reactions (e.g., conjugation reactions), the products of which are final toxicants. We identified a high level of activation for three groups of compounds (General Chemicals, Drugs, and Natural Products) yielding activated metabolites and the generally low participation of Physiological Compounds in bioactivation reactions. In the group of General Chemicals, P450 enzymes 1A1, 1A2, and 1B1 dominate in the formation of activated metabolites.Drugs are mostly activated by the enzyme P450 3A4, and Natural Products by P450s 1A2, 2E1, and 3A4. Physiological Compounds showed no clearly dominant enzyme, but the highest numbers of activations are attributed to P450 1A, 1B1, and 3A enzymes. The results thus show, perhaps not surprisingly, that Physiological Compounds are infrequent substrates in bioactivation reactions catalyzed by P450 enzyme Families 1-4, with the exception of estrogens and arachidonic acid. The results thus provide information on the enzymes that activate specific groups of chemicals to toxic metabolites.

Identification of CYP 2A6 inhibitors in an effort to mitigate the harmful effects of the phytochemical nicotine.

Aim:In this study, our goal was to study the inhibition of nicotine metabolism by P450 2A6, as a means for reduction in tobacco use and consequently the prevention of smoking-related cancers. Nicotine, a phytochemical, is an addictive stimulant, responsible for the tobacco-dependence in smokers. Many of the other phytochemicals in tobacco, including polycyclic aromatic hydrocarbons, N-nitrosamines, and aromatic amines, are potent systemic carcinogens. Tobacco smoking causes about one of every five deaths in the United States annually. Nicotine plasma concentration is maintained by the smokers’ smoking behavior within a small range. Nicotine is metabolized by cytochrome P450s 2A6 and 2A13 to cotinine. This metabolism causes a decrease in nicotine plasma levels, which in turn leads to increased tobacco smoking, and increased exposure to the tobacco carcinogens.Methods:Using the phytochemical nicotine as a lead structure, and taking its interactions with the P450 2A6 binding pocket into consideration, new pyridine derivatives were designed and synthesized as potential selective mechanism-based inhibitors for this enzyme.Results:The design and synthesis of two series of novel pyridine-based compounds, with varying substituents and substitution locations on the pyridine ring, as well as their inhibitory activities on cytochrome P450 2A6 and their interactions with its active site are discussed here. Substitutions at position 3 of the pyridine ring with an imidazole or propargyl ether containing group showed the most optimal interactions with the P4502A6 active site.Conclusion:The pyridine compounds with an imidazole or propargyl ether containing substituent on position 3 were found to be promising lead compounds for further development. Hydrogen-bonding interactions were determined to be crucial for effective binding of these molecules within the P450 2A6 active site.

Human cytochrome P450 enzymes bind drugs and other substrates mainly through conformational-selection modes

Cytochrome P450 (P450) enzymes are major catalysts involved in the oxidations of most drugs, steroids, carcinogens, fat-soluble vitamins, and natural products. The binding of substrates to some of the 57 human P450s and other mammalian P450s is more complex than a two-state system and has been proposed to involve mechanisms such as multiple ligand occupancy, induced-fit, and conformational-selection. Here, we used kinetic analysis of binding with multiple concentrations of substrates and computational modeling of these data to discern possible binding modes of several human P450s. We observed that P450 2D6 binds its ligand rolapitant in a mechanism involving conformational-selection. P450 4A11 bound the substrate lauric acid via conformational-selection, as did P450 2C8 with palmitic acid. Binding of the steroid progesterone to P450 21A2 was also best described by a conformational-selection model. Hexyl isonicotinate binding to P450 2E1 could be described by either a conformational-selection or an induced-fit model. Simulation of the binding of the ligands midazolam, bromocriptine, testosterone, and ketoconazole to P450 3A4 was consistent with an induced-fit or a conformational-selection model, but the concentration dependence of binding rates for varying both P450 3A4 and midazolam concentrations revealed discordance in the parameters, indicative of conformational-selection. Binding of the P450s 2C8, 2D6, 3A4, 4A11, and 21A2 was best described by conformational-selection, and P450 2E1 appeared to fit either mode. These findings highlight the complexity of human P450-substrate interactions and that conformational-selection is a dominant feature of many of these interactions.

Identification and Characterization of a Selective Human Carbonyl Reductase 1 Substrate.

During drug discovery efforts targeting inhibition of cytochrome P450 11B2 (CYP11B2)-mediated production of aldosterone as a therapeutic approach for the treatment of chronic kidney disease and hypertension, (S)-6-(5-fluoro-4-(1-hydroxyethyl)pyridin-3-yl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxamide (1) was identified as a potent and selective inhibitor of CYP11B2. Preclinical studies characterized 1 as low clearance in both in vitro test systems and in vivo in preclinical species. Despite low metabolic conversion, an active ketone metabolite (2) was identified from in vitro metabolite-identification studies. Due to the inhibitory activity of 2 against CYP11B2 as well as the potential for it to undergo reductive metabolism back to 1, the formation and elimination of 2 were characterized and are the focus of this manuscript. A series of in vitro investigations determined that 1 was slowly oxidized to 2 by cytochrome P450s 2D6, 3A4, and 3A5, followed by stereoselective reduction back to 1 and not its enantiomer (3). Importantly, reduction of 2 was mediated by an NADPH-dependent cytosolic enzyme. Studies with human cytosolic fractions from multiple tissues, selective inhibitors, and recombinantly expressed enzymes indicated that carbonyl reductase 1 (CBR1) is responsible for this transformation in humans. Carbonyl reduction is emerging as an important pathway for endogenous and xenobiotic metabolism. With a lack of selective substrates and inhibitors to enable characterization of the involvement of CBR1, 2 could be a useful probe to assess CBR1 activity in vitro in both subcellular fractions and in cell-based systems.

Sulfenylation of Human Liver and Kidney Microsomal Cytochromes P450 and Other Drug-Metabolizing Enzymes as a Response to Redox Alteration

The lumen of the endoplasmic reticulum (ER) provides an oxidizing environment to aid in the formation of disulfide bonds, which is tightly regulated by both antioxidant proteins and small molecules. On the cytoplasmic side of the ER, cytochrome P450 (P450) proteins have been identified as a superfamily of enzymes that are important in the formation of endogenous chemicals as well as in the detoxication of xenobiotics. Our previous report described oxidative inhibition of P450 Family 4 enzymes via oxidation of the heme-thiolate cysteine to a sulfenic acid (-SOH) (Albertolle, M. E. et al. (2017) J. Biol. Chem. 292, 11230-11242). Further proteomic analyses of murine kidney and liver microsomes led to the finding that a number of other drug-metabolizing enzymes located in the ER are also redox-regulated in this manner. We expanded our analysis of sulfenylated enzymes to human liver and kidney microsomes. Evaluation of the sulfenylation, catalytic activity, and spectral properties of P450s 1A2, 2C8, 2D6, and 3A4 led to the identification of two classes of redox sensitivity in P450 enzymes: heme-thiolate-sensitive and thiol-insensitive. These findings provide evidence for a mammalian P450 regulatory mechanism, which may also be relevant to other drug-metabolizing enzymes. (Data are available via ProteomeXchange with identifier PXD007913.).

Transient Expression of Human Cytochrome P450s 2D6 and 3A4 in Nicotiana benthamiana Provides a Possibility for Rapid Substrate Testing and Production of Novel Compounds.

Employment of transient expression of foreign genes for bioconversion of pharmaceutically valuable low-molecular-weight compounds, including plant secondary metabolites, is an enticing trend still scantily explored in plant biotechnology. In the present work, an efficient protocol for rapid assessment of synthetic and plant-derived metabolites as potential substrates for human P450s (CYP2D6 and CYP3A4) via Agrobacterium-mediated transient expression in Nicotiana benthamiana is put forth. Animal P450s with broad substrate specificity are promising candidates for transformation of diverse metabolites. The efficiency of P450s in heterologous surroundings is not always satisfactory and depends on the availability of an associated electron-transfer enzyme. Plants represent an attractive assortment of prospective hosts for foreign P450s expression. The optimal composition of genetic blocks providing the highest transient expression efficiency is designed, an effective substrate administration scheme is validated, and biological activity of the investigated P450s against loratadine and several indole alkaloids with different molecular scaffold structures is tested. A novel indole alkaloid, 11-hydroxycorynanthine, is isolated from N. benthamiana plants transiently expressing CYP2D6 and supplemented with corynanthine, and its structure was elucidated. The proposed technique might be of value in realization of combinatorial biosynthesis concept comprising the junction of heterologous enzymes and substrates in different metabolic surroundings.

Structural differences exhibited by cytochrome P450S 3A5 and 3A4

Structural differences exhibited by cytochrome P450S 3A5 and 3A4

The X-Ray Crystal Structure of the Human Mono-Oxygenase Cytochrome P450 3A5-Ritonavir Complex Reveals Active Site Differences between P450s 3A4 and 3A5.

The contributions of cytochrome P450 3A5 to the metabolic clearance of marketed drugs is unclear, but its probable role is to augment the metabolism of several drugs that are largely cleared by P450 3A4. Selective metabolism by 3A4 is often a concern in drug development owing to potential drug-drug interactions and the variability of 3A4 and 3A5 expression. The contribution of P450 3A5 to these clearance pathways varies between individuals owing to genetic differences and similarities and differences in the metabolic properties of 3A5 compared with 3A4. To better understand the structural differences between P450s 3A4 and 3A5, the structure of 3A5 complexed with ritonavir was determined by X-ray crystallography to a limiting resolution of 2.91 Å. The secondary and tertiary structures of 3A5 and 3A4 are similar, but the architectures of their active sites differ. The 3A5 active site is taller and narrower than that of 3A4. As a result, ritonavir adopts a distinctly different conformation to fit into the cavity of 3A5 than seen for 3A4. These structural changes reflect amino acid differences that alter the conformation of the helix F through helix G region in the upper portion of the cavity and ionic interactions between residues in the beta-sheet domain that reduce the width of the cavity. The structural differences exhibited by 3A4 and 3A5 suggest that the overlap of catalytic activities may reflect molecular flexibility that determines how alternative conformers fit into the different active site architectures of the two enzymes.

Hepatic expression of cytochrome P450 enzymes in non-human primate species.

Cytochromes P450 (P450) largely remain to be characterized in great apes. Comparative immunochemical detection of drug metabolizing forms of P450s 1A, 2A, 2B, 2C, 2D, 2E, 2J, 3A, 4A, and 4F in liver microsomes from chimpanzees, gorillas, orangutans, gibbons, cynomolgus and rhesus macaques, and common marmosets were carried out.

REDX08608, a Novel, Potent and Selective, Reversible BTK Inhibitor with Efficacy and Equivalent Potency Against Wild-Type and Mutant C481S BTK

Here we report the preclinical profile of REDX08608 our novel, potent and selective, reversible BTK inhibitor that is equipotent against wild-type and mutant C481S BTK.Bruton's tyrosine kinase (BTK) is a member of the src-related Tec family of cytoplasmic tyrosine kinases and plays a key role in the BCR signaling pathway, which is required for the development, activation and survival of B-cells. BTK is a clinically validated target to treat B-cell malignancies that are dependent on BCR signaling i.e.CLL and NHL with ibrutinib approved for the treatment of CLL, MCL and WM. Irreversible and covalent reversible BTK inhibitors such as ibrutinib, acalabrutinib and GS-4059 specifically target a cysteine residue C481 within BTK and mutations at this site clearly interfere with covalent drug binding. C481S, C481Y, C481R, C481F mutations have been reported and linked to cases of resistance that have emerged in patients with CLL progression following treatment with ibrutinib (Byrd2016, Inhye2016, Maddocks2015, Woaych2014). Redx reversible BTK inhibitor, REDX08608, aims to overcome this resistance mechanism by targeting both wild type and C481-mutated BTK. Redx have recently presented REDX06961 our BTK probe (Guisot2016, AACR#4795) and, following lead optimization, we are now disclosing REDX08608, our lead compound, a potent, reversible and selective BTK inhibitor, which displays an improved profile including superior pharmacokinetics.REDX08608, reversibly, inhibits WT and C481S BTK and displays nanomolar binding affinity and potency in biochemical and cellular-based assays.REDX08608 inhibits BTK signaling and growth in cell lines dependent on the BTK pathway such the OCI-LY10 ABC-DLBCL cell line. Importantly, REDX08608 also inhibits BTK signaling in primary CLL cells. In human whole blood and isolated human PBMCs, REDX08608 inhibits activation of B-cells at nanomolar concentrations measured by inhibition of immunoglobulin-induced CD69 in CD19+cells.REDX08608 is highly selective when tested against a panel of 468 kinases and exhibits improved target specificity with >100-fold selectivity against other Tec and Src kinase family members (ITK, TXK, BMX, TEC, BLK, CSK, FYN, HCK, LCK, SRC) and >400-fold selectivity against EGFR.REDX08608 was fully profiled through DMPK in vitro assays including metabolic stability, plasma stability, cytochrome P450 inhibition, PXR activation/cytochrome P450 activity, time dependent inhibition and cytochrome P450 reaction phenotyping. REDX08608 was shown to have an acceptable metabolic and plasma stability profile across species (mouse, rat, dog, monkey and human). REDX08608 displayed no evidence of PXR activation or time dependent inhibition. IC50s were determined for human cytochrome P450s (1A2, 2D6, 2C9, 2C19 and 3A4) and were all >10 µM. Good exposure, oral bioavailability and half-life were demonstrated for REDX08608 in mouse, rat and dog, with dose linearity assessment performed in mouse (F = 73-100%, CL= 11% liver blood flow in mice; F = 55-84%, CL = 28% liver blood flow in rat; F = 85%, CL= 10% liver blood flow in dog). In vivo efficacy studies in preclinical models will also be disclosed.In conclusion, REDX08608 is a potent and selective, reversible BTK inhibitor with efficacy in lymphoma cell lines that offers the potential to target both wild-type BTK and an important emerging resistance mechanism in patients with CLL progression following ibrutinib-treatment. DisclosuresGuisot:Redx Oncology Ltd - Redx Pharma Plc: Employment. Best:Redx Oncology Ltd - Redx Pharma Plc: Employment. Wright:Redx Oncology Ltd - Redx Pharma Plc: Employment. Thomason:Redx Oncology Ltd - Redx Pharma Plc: Employment. Woyach:Acerta: Research Funding; Karyopharm: Research Funding; Morphosys: Research Funding. Abet:Redx Oncology Ltd - Redx Pharma Plc: Employment. Castagna:Redx Oncology Ltd - Redx Pharma Plc: Employment. Cousin:Redx Oncology Ltd - Redx Pharma Plc: Employment. Emmerich:Redx Oncology Ltd - Redx Pharma Plc: Employment. Ho:Redx Oncology Ltd - Redx Pharma Plc: Employment. Kelly:Redx Oncology Ltd - Redx Pharma Plc: Employment. King-Tours:Redx Oncology Ltd - Redx Pharma Plc: Employment. Lyons:Redx Oncology Ltd - Redx Pharma Plc: Employment. Muller:Redx Oncology Ltd - Redx Pharma Plc: Employment. Refuerzo:Redx Oncology Ltd - Redx Pharma Plc: Employment. Sargent:Redx Oncology Ltd - Redx Pharma Plc: Employment. Talab:Redx Oncology Ltd - Redx Pharma Plc: Employment. Bingham:Redx Oncology Ltd - Redx Pharma Plc: Employment. Phillips:Redx Oncology Ltd - Redx Pharma Plc: Employment. Armer:Redx Oncology Ltd - Redx Pharma Plc: Employment.

Construction of Metabolism Prediction Models for CYP450 3A4, 2D6, and 2C9 Based on Microsomal Metabolic Reaction System.

During the past decades, there have been continuous attempts in the prediction of metabolism mediated by cytochrome P450s (CYP450s) 3A4, 2D6, and 2C9. However, it has indeed remained a huge challenge to accurately predict the metabolism of xenobiotics mediated by these enzymes. To address this issue, microsomal metabolic reaction system (MMRS)—a novel concept, which integrates information about site of metabolism (SOM) and enzyme—was introduced. By incorporating the use of multiple feature selection (FS) techniques (ChiSquared (CHI), InfoGain (IG), GainRatio (GR), Relief) and hybrid classification procedures (Kstar, Bayes (BN), K-nearest neighbours (IBK), C4.5 decision tree (J48), RandomForest (RF), Support vector machines (SVM), AdaBoostM1, Bagging), metabolism prediction models were established based on metabolism data released by Sheridan et al. Four major biotransformations, including aliphatic C-hydroxylation, aromatic C-hydroxylation, N-dealkylation and O-dealkylation, were involved. For validation, the overall accuracies of all four biotransformations exceeded 0.95. For receiver operating characteristic (ROC) analysis, each of these models gave a significant area under curve (AUC) value >0.98. In addition, an external test was performed based on dataset published previously. As a result, 87.7% of the potential SOMs were correctly identified by our four models. In summary, four MMRS-based models were established, which can be used to predict the metabolism mediated by CYP3A4, 2D6, and 2C9 with high accuracy.

Identification, Synthesis, and Biological Evaluation of the Major Human Metabolite of NLRP3 Inflammasome Inhibitor MCC950.

MCC950 is an orally bioavailable small molecule inhibitor of the NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome that exhibits remarkable activity in multiple models of inflammatory disease. Incubation of MCC950 with human liver microsomes, and subsequent analysis by HPLC-MS/MS, revealed a major metabolite, where hydroxylation of MCC950 had occurred on the 1,2,3,5,6,7-hexahydro-s-indacene moiety. Three possible regioisomers were synthesized, and coelution using HPLC-MS/MS confirmed the structure of the metabolite. Further synthesis of individual enantiomers and coelution studies using a chiral column in HPLC-MS/MS showed the metabolite was R-(+)- N-((1-hydroxy-1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl)-4-(2-hydroxypropan-2-yl)furan-2-sulfonamide (2a). Incubation of MCC950 with a panel of cytochrome P450 enzymes showed P450s 2A6, 2C9, 2C18, 2C19, 2J2, and 3A4 catalyze the formation of the major metabolite 2a, with a lower level of activity shown by P450s 1A2 and 2B6. All of the synthesized compounds were tested for inhibition of NLRP3-induced production of the pro-inflammatory cytokine IL-1β from human monocyte derived macrophages. The identified metabolite 2a was 170-fold less potent than MCC950, while one regioisomer had nanomolar inhibitory activity. These findings also give first insight into the SAR of the hexahydroindacene moiety.

Structure-Function Studies of Naphthalene, Phenanthrene, Biphenyl, and Their Derivatives in Interaction with and Oxidation by Cytochromes P450 2A13 and 2A6.

Naphthalene, phenanthrene, biphenyl, and their derivatives having different ethynyl, propynyl, butynyl, and propargyl ether substitutions were examined for their interaction with and oxidation by cytochromes P450 (P450) 2A13 and 2A6. Spectral interaction studies suggested that most of these chemicals interacted with P450 2A13 to induce Type I binding spectra more readily than with P450 2A6. Among the various substituted derivatives examined, 2-ethynylnaphthalene, 2-naphthalene propargyl ether, 3-ethynylphenanthrene, and 4-biphenyl propargyl ether had larger ΔAmax/Ks values in inducing Type I binding spectra with P450 2A13 than their parent compounds. P450 2A13 was found to oxidize naphthalene, phenanthrene, and biphenyl to 1-naphthol, 9-hydroxyphenanthrene, and 2- and/or 4-hydroxybiphenyl, respectively, at much higher rates than P450 2A6. Other human P450 enzymes including P450s 1A1, 1A2, 1B1, 2C9, and 3A4 had lower rates of oxidation of naphthalene, phenanthrene, and biphenyl than P450s 2A13 and 2A6. Those alkynylated derivatives that strongly induced Type I binding spectra with P450s 2A13 and 2A6 were extensively oxidized by these enzymes upon analysis with HPLC. Molecular docking studies supported the hypothesis that ligand-interaction energies (U values) obtained with reported crystal structures of P450 2A13 and 2A6 bound to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, indole, pilocarpine, nicotine, and coumarin are of use in understanding the basis of possible molecular interactions of these xenobiotic chemicals with the active sites of P450 2A13 and 2A6 enzymes. In fact, the ligand-interaction energies with P450 2A13 4EJG bound to these chemicals were found to relate to their induction of Type I binding spectra.

Role of SQSTM1/p62 in Cytochrome P450 Autophagy?

We previously reported that primary hepatocytes depleted of heme upon N‐methyl‐protoporphyrin (NMPP)‐treatment, exhibit a rapid loss of P450s 3A and 2B. We now document that protoporphyrin IX (PPIX)‐ as well as Zinc‐protoporphyrin IX (ZnPP)‐treated hepatocytes, cellular models of acute hepatic protoporphyria and/or oxidative stress, showed an even greater loss of CYP2B. This loss is associated with a significant oligomerization and crosslinking of SQSTM1/p62, a cellular protein scaffold that selects substrate‐cargo for autophagy. This CYP2B loss can also be partially reversed by the autophagic/lysosomal inhibitors 3‐methyladenine and NH4Cl. These observations suggest that p62 mediates the autophagic‐lysosomal degradation of CYP2B. To test this possibility, we co‐expressed p62 together with CYP2B6 and CYP3A4 in HEK297T cells. To our surprise, under normal conditions, p62 co‐expression significantly increased CYP2B6 as well as CYP3A4 protein levels. Co‐immunoprecipitation analyses demonstrated that CYP2B6 and p62 physically interacted with each other. p62‐Myc‐pull‐down assays from HepG2 cells and HEK293T cells overexpressing p62‐Myc, followed by LC‐MS/MS proteomic analysis also revealed appreciable endogenous CYP3A presences, thereby supporting their stable intracellular protein‐protein interaction. A p62‐truncation mutant without the ubiquitin‐binding domain (UBA) also interacted with CYP2B, suggesting that this interaction does not depend on CYP2B ubiquitination; serial p62‐truncation analyses revealed a previously unidentified p62‐domain to be responsible for CYP2B‐protein interaction. Additional studies in primary p62−/−‐mouse hepatocytes revealed a significant decrease of CYP2B levels upon phenobarbital induction. Collectively, these findings suggest a novel p62 working‐model: p62 binds protein substrates (CYP2B6) under normal/basal conditions and stabilizes them, whereas under stressful (PPIX, ZnPPIX) conditions, it shuttles the possibly damaged proteins to their autophagic‐lysosomal degradation. Studies are currently in progress in atg5−/− autophagy‐deficient MEF cells to test this hypothetic model.Support or Funding InformationSupported by NIH grants DK26506, GM44037 and DK26743 (Liver Center).