Characterization of a Nitro-Forming Enzyme Involved in Fosfazinomycin Biosynthesis.

N-hydroxylating monooxygenases (NMOs) are a subclass of flavin-dependent enzymes that hydroxylate nitrogen atoms. Recently, unique NMOs that perform multiple reactions on one substrate molecule have been identified. Fosfazinomycin M (FzmM) is one such NMO, forming nitrosuccinate from aspartate (Asp) in the fosfazinomycin biosynthetic pathway in some Streptomyces sp. This work details the biochemical and kinetic analysis of FzmM. Steady-state kinetic investigation shows that FzmM performs a coupled reaction with Asp (kcat, 3.0 ± 0.01 s-1) forming nitrosuccinate, which can be converted to fumarate and nitrite by the action of FzmL. FzmM displays a 70-fold higher kcat/KM value for NADPH compared to NADH and has a narrow optimal pH range (7.5-8.0). Contrary to other NMOs where the kred is rate-limiting, FzmM exhibits a very fast kred (50 ± 0.01 s-1 at 4 °C) with NADPH. NADPH binds at a KD value of ∼400 µM, and hydride transfer occurs with pro-R stereochemistry. Oxidation of FzmM in the absence of Asp exhibits a spectrum with a shoulder at ∼370 nm, consistent with the formation of a C(4a)-hydroperoxyflavin intermediate, which decays into oxidized flavin and hydrogen peroxide at a rate 100-fold slower than the kcat. This reaction is enhanced in the presence of Asp with a slightly faster kox than the kcat, suggesting that flavin dehydration or Asp oxidation is partially rate limiting. Multiple sequence analyses of FzmM to NMOs identified conserved residues involved in flavin binding but not for NADPH. Additional sequence analysis to related monooxygenases suggests that FzmM shares sequence motifs absent in other NMOs.

Contrasting Local and Macroscopic Effects of Collagen Hydroxylation.

Collagen is heavily hydroxylated. Experiments show that proline hydroxylation is important to triple helix (monomer) stability, fibril assembly, and interaction of fibrils with other molecules. Nevertheless, experiments also show that even without hydroxylation, type I collagen does assemble into its native D-banded fibrillar structure. This raises two questions. Firstly, even though hydroxylation removal marginally affects macroscopic structure, how does such an extensive chemical change, which is expected to substantially reduce hydrogen bonding capacity, affect local structure? Secondly, how does such a chemical perturbation, which is expected to substantially decrease electrostatic attraction between monomers, affect collagen's mechanical properties? To address these issues, we conduct a benchmarked molecular dynamics study of rat type I fibrils in the presence and absence of hydroxylation. Our simulations reproduce the experimental observation that hydroxylation removal has a minimal effect on collagen's D-band length. We also find that the gap-overlap ratio, monomer width and monomer length are minimally affected. Surprisingly, we find that de-hydroxylation also has a minor effect on the fibril's Young's modulus, and elastic stress build up is also accompanied by tightening of triple-helix windings. In terms of local structure, de-hydroxylation does result in a substantial drop (23%) in inter-monomer hydrogen bonding. However, at the same time, the local structures and inter-monomer hydrogen bonding networks of non-hydroxylated amino acids are also affected. It seems that it is this intrinsic plasticity in inter-monomer interactions that preclude fibrils from undergoing any large changes in macroscopic properties. Nevertheless, changes in local structure can be expected to directly impact collagen's interaction with extra-cellular matrix proteins. In general, this study highlights a key challenge in tissue engineering and medicine related to mapping collagen chemistry to macroscopic properties but suggests a path forward to address it using molecular dynamics simulations.

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 ß 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.

Cytochrome P450-Catalyzed Metabolism of Cannabidiol to the Active Metabolite 7-Hydroxy-Cannabidiol.

Cannabidiol (CBD) is a naturally occurring nonpsychotoxic phytocannabinoid that has gained increasing attention as a popular consumer product and for its use in Food and Drug Administration-approved Epidiolex (CBD oral solution) for the treatment of Lennox-Gastaut syndrome and Dravet syndrome. CBD was previously reported to be metabolized primarily by CYP2C19 and CYP3A4, with minor contributions from UDP-glucuronosyltransferases. 7-Hydroxy-CBD (7-OH-CBD) is the primary active metabolite with equipotent activity compared with CBD. Given the polymorphic nature of CYP2C19, we hypothesized that variable CYP2C19 expression may lead to interindividual differences in CBD metabolism to 7-OH-CBD. The objectives of this study were to further characterize the roles of cytochrome P450 enzymes in CBD metabolism, specifically to the active metabolite 7-OH-CBD, and to investigate the impact of CYP2C19 polymorphism on CBD metabolism in genotyped human liver microsomes. The results from reaction phenotyping experiments with recombinant cytochrome P450 enzymes and cytochrome P450-selective chemical inhibitors indicated that both CYP2C19 and CYP2C9 are capable of CBD metabolism to 7-OH-CBD. CYP3A played a major role in CBD metabolic clearance via oxidation at sites other than the 7-position. In genotyped human liver microsomes, 7-OH-CBD formation was positively correlated with CYP2C19 activity but was not associated with CYP2C19 genotype. In a subset of single-donor human liver microsomes with moderate to low CYP2C19 activity, CYP2C9 inhibition significantly reduced 7-OH-CBD formation, suggesting that CYP2C9 may play a greater role in CBD 7-hydroxylation than previously thought. Collectively, these data indicate that both CYP2C19 and CYP2C9 are important contributors in CBD metabolism to the active metabolite 7-OH-CBD. SIGNIFICANCE STATEMENT: This study demonstrates that both CYP2C19 and CYP2C9 are involved in CBD metabolism to the active metabolite 7-OH-CBD and that CYP3A4 is a major contributor to CBD metabolism through pathways other than 7-hydroxylation. 7-OH-CBD formation was associated with human liver microsomal CYP2C19 activity, but not CYP2C19 genotype, and CYP2C9 was found to contribute significantly to 7-OH-CBD generation. These findings have implications for patients taking CBD who may be at risk for clinically important cytochrome P450-mediated drug interactions.

Functional Characterization of 40 CYP3A4 Variants by Assessing Midazolam 1'-Hydroxylation and Testosterone 6ß-Hydroxylation.

CYP3A4 is among the most abundant liver and intestinal drug-metabolizing cytochrome P450 enzymes, contributing to the metabolism of more than 30% of clinically used drugs. Therefore, interindividual variability in CYP3A4 activity is a frequent cause of reduced drug efficacy and adverse effects. In this study, we characterized wild-type CYP3A4 and 40 CYP3A4 variants, including 11 new variants, detected among 4773 Japanese individuals by assessing CYP3A4 enzymatic activities for two representative substrates (midazolam and testosterone). The reduced carbon monoxide-difference spectra of wild-type CYP3A4 and 31 CYP3A4 variants produced with our established mammalian cell expression system were determined by measuring the increase in maximum absorption at 450 nm after carbon monoxide treatment. The kinetic parameters of midazolam and testosterone hydroxylation by wild-type CYP3A4 and 29 CYP3A4 variants (K m , k cat , and catalytic efficiency) were determined, and the causes of their kinetic differences were evaluated by three-dimensional structural modeling. Our findings offer insight into the mechanism underlying interindividual differences in CYP3A4-dependent drug metabolism. Moreover, our results provide guidance for improving drug administration protocols by considering the information on CYP3A4 genetic polymorphisms. SIGNIFICANCE STATEMENT: CYP3A4 metabolizes more than 30% of clinically used drugs. Interindividual differences in drug efficacy and adverse-effect rates have been linked to ethnicity-specific differences in CYP3A4 gene variants in Asian populations, including Japanese individuals, indicating the presence of CYP3A4 polymorphisms resulting in the increased expression of loss-of-function variants. This study detected alterations in CYP3A4 activity due to amino acid substitutions by assessing the enzymatic activities of coding variants for two representative CYP3A4 substrates.

Effects of acid and lactone forms of statins on S-warfarin 7-hydroxylation catalyzed by human liver microsomes and recombinant CYP2C9 variants (CYP2C9.1 and CYP2C9.3).

The inhibition of CYP2C9-mediated warfarin metabolism by acid or lactone forms of statin converted in the body and effects of CYP2C9 genetic variants on their inhibition are not fully understood. Here, the effects of acid and lactone forms of statins on S-warfarin 7-hydroxylation were investigated in vitro. S-Warfarin 7-hydroxylase activities of human liver microsomes (HLMs), recombinant CYP2C9.1 (rCYP2C9.1), and rCYP2C9.3 (Ile359Leu variant) in the presence of statins were determined by high-performance liquid chromatography. Lactone forms of atorvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin inhibited the activity of HLMs more potently than the corresponding acid forms, whereas fluvastatin acid showed stronger inhibition than fluvastatin lactone. When the effects of statins on rCYP2C9 variants were examined, inhibition profiles of acid versus lactone forms of statins except for fluvastatin were similar between rCYP2C9.1 and rCYP2C9.3. However, the degrees of inhibition by atorvastatin lactone, fluvastatin acid, fluvastatin lactone, lovastatin lactone, and pitavastatin lactone (Ki values) were significantly different between these variants. These results indicated that lactone forms of statins other than fluvastatin showed more potent inhibition of CYP2C9-catalyzed S-warfarin 7-hydroxylation than the corresponding acid forms. Furthermore, our results indicated that Ile359Leu substitution in CYP2C9 affected the inhibitory potencies of statins.

Role of tetrachloro-1,4-benzoquinone reductase in phenylalanine hydroxylation system and pentachlorophenol degradation in Bacillus cereus AOA-CPS1.

This study reports a ≅12.5 kDa protein tetrachloro-1,4-benzoquinone reductase (CpsD) from Bacillus cereus strain AOA-CPS1 (BcAOA). CpsD is purified to homogeneity with a total yield of 35% and specific activity of 160 U·mg-1 of protein. CpsD showed optimal activity at pH 7.5 and 40 °C. The enzyme was found to be functionally stable between pH 7.0-7.5 and temperature between 30 °C and 35 °C. CpsD activity was enhanced by Fe2+ and inhibited by sodium azide and SDS. CpsD followed Michaelis-Menten kinetic exhibiting an apparent vmax, Km, kcat and kcat/Km values of 0.071 µmol·s-1, 94 µmol, 0.029 s-1 and 3.13 × 10-4 s-1·µmol-1, respectively, for substrate tetrachloro-1,4-benzoquinone. The bioinformatics analysis indicated that CpsD belongs to the PCD/DCoH superfamily, with specific conserved protein domains of pterin-4α-carbinolamine  dehydratase (PCD). This study proposed that CpsD catalysed the reduction of tetrachloro-1,4-benzoquinone to tetrachloro-p-hydroquinone and released the products found in phenylalanine hydroxylation system (PheOHS) via a Ping-Pong or atypical ternary mechanism; and regulate expression of phenylalanine 4-monooxygenase by blocking reverse flux in BcAOA PheOHS using a probable Yin-Yang mechanism. The study also concluded that CpsD may play a catalytic and regulatory role in BcAOA PheOHS and pentachlorophenol degradation pathway.

LC-Q-TOF/MS-oriented systemic metabolism study of pedunculoside with in vitro and in vivo biotransformation.

As a triterpene saponin, pedunculoside is one of the most abundant, representative and active components in plants of genus Ilex (Aquifoliaceae). Pedunculoside has been used to treat myocardial ischemia, ameliorate hyperlipidemia and prevent liver injury. In this paper, a systemic in vitro liver microsomes / S9 and intestinal bacteria incubation, and in vivo animal experiment were performed, using LC-Q-TOF/MS analysis and a three-step data processing protocol. As a result, Bifidobacterium adolescentis and Bifidobacterium breve were identified to potentially metabolize pedunculoside among the intestinal bacteria tested. A total of 11 metabolites were found and tentatively identified, with 6 in both microsomal and bacterial incubation systems, and 9 after rats orally administered with pedunculoside. The metabolites detected involving both phase I and phase II metabolism, mainly through deglycosylation (hydrolyzation), dehydrogenation, hydroxylation and conjugation, and some of them underwent more than one-step metabolic reactions. Most of the metabolites have not been reported before. In vitro, liver microsome and intestinal bacteria prefer to metabolize pedunculoside in totally different ways; while in vivo, intestinal tract is the most important site for the metabolism and excretion of pedunculoside, where both intestinal bacteria and the host metabolic enzymes participate in its metabolism and disposition. The importance of intestinal bacteria should be highlighted. This study would contribute to a better understanding of pedunculoside metabolism, which can provide scientific evidence for its pharmacodynamic mechanism research and prove its clinical application.

In vitro studies on the metabolism of saikogenins and the detection of their metabolites in authentic biosamples.

Saikosaponins are the representative bioactive ingredient in the Radix Bupleuri. Previous studies have reported that saikosaponins are prone to losing their glycones and being converted to corresponding prosaikogenins and saikogenins by intestinal bacteria. However, the microsomal cytochrome P450-mediated metabolism of these deglycosylated metabolites is still unknown. This research performed in vitro studies of five saikogenins in rat and human liver microsomes. High-performance liquid chromatography coupled with a hybrid ion trap and time-of-flight mass spectrometry (HPLC-IT/TOF-MS) was employed to identify the metabolites. To confirm the metabolites detected in vitro, plasma and feces obtained from rats administrating several saikogenins were also analyzed by high-performance liquid chromatography with triple-quadrupole mass spectrometry (HPLC-QqQ-MS). The in vitro metabolic stability of saikogenins was ranked as follows: SGF＞SGG＞SGE＞SGA＞SGD. A total of 71 metabolites generated by hydroxylation, carboxylation, and dehydrogenation, as well as combinations of these steps, were identified by accurate mass measurement and MSn fragmentation behavior. Among eight metabolic pathways, monohydroxylation or carboxylation was the major metabolic pathway both in vitro and in vivo. Analysis of in vivo biological samples suggested that analytical targets for saikogenins should be the compounds themselves and their oxidized metabolites. This research provides a basis for further studies of the in vivo metabolism of saikosaponins in humans.

Stereoselective Oxidation Kinetics of Deoxycholate in Recombinant and Microsomal CYP3A Enzymes: Deoxycholate 19-Hydroxylation Is an In Vitro Marker of CYP3A7 Activity.

The primary bile acids (BAs) synthesized from cholesterol in the liver are converted to secondary BAs by gut microbiota. It was recently disclosed that the major secondary BA, deoxycholate (DCA) species, is stereoselectively oxidized to tertiary BAs exclusively by CYP3A enzymes. This work subsequently investigated the in vitro oxidation kinetics of DCA at C-1ß, C-3ß, C-4ß, C-5ß, C-6α, C-6ß, and C-19 in recombinant CYP3A enzymes and naive enzymes in human liver microsomes (HLMs). The stereoselective oxidation of DCA fit well with Hill kinetics at 1-300 µM in both recombinant CYP3A enzymes and pooled HLMs. With no contributions or trace contributions from CYP3A5, CYP3A7 favors oxidation at C-19, C-4ß, C-6α, C-3ß, and C-1ß, whereas CYP3A4 favors the oxidation at C-5ß and C-6ß compared with each other. Correlation between DCA oxidation and testosterone 6ß-hydroxylation in 14 adult single-donor HLMs provided proof-of-concept evidence that DCA 19-hydroxylation is an in vitro marker reaction for CYP3A7 activity, whereas oxidation at other sites represents mixed indicators for CYP3A4 and CYP3A7 activities. Deactivation caused by DCA-induced cytochrome P450-cytochrome P420 conversion, as shown by the spectral titrations of isolated CYP3A proteins, was observed when DCA levels were near or higher than the critical micelle concentration (about 1500 µM). Unlike CYP3A4, CYP3A7 showed abnormally elevated activities at 500 and 750 µM, which might be associated with an altered affinity for DCA multimers. The disclosed kinetic and functional roles of CYP3A isoforms in disposing of the gut bacteria-derived DCA may help in understanding the structural and functional mechanisms of CYP3A.

Atipamezole is a promising non-discriminative inhibitor against pan-CYP450 including diclofenac 4'-hydroxylation: A comparison with ABT for drug ADME optimization and mechanism study.

1-aminobenzotriazole (ABT) is a known P450 enzyme non-selective inactivator that serves as a tool for assessing P450-mediated metabolism. However, many findings demonstrated that ABT was ineffective with human CYP2C9. A profound pan-CYP450 inhibitor is desired avoid the risk of incomplete inhibition of P450, especially CYP2C9. Atipamezole is commonly used to recover animals from sedation-anesthesia induced by α2-adrenoceptor agonists. The purpose of this study is to evaluate atipamezole as a non-selective inhibitor of P450 enzymes and compare it with ABT. Inhibition toward seven major human CYP450 isoform was determined for atipamezole and ABT in human, rat, and dog liver microsomes for the direct and time-dependent inhibition potentials. IC50 values toward human and animal CYPs without preincubation are 0.02-7.93 µM and 20.9-1798 µM for atipamezole and ABT, respectively. The IC50 values of ABT after preincubation shift to 4.06-460 µM. Atipamezole has more effective inhibition to CYP2C9 mediated diclofenac hydroxylation in human and animal liver microsomes with IC50 values of 1.50-5.20 µM than that of ABT at 74.7-460 µM. No IC50 shift was observed for atipamezole to CYP isoforms. In vivo utility of atipamezole was assessed by co-dosing with diclofenac in rats. At 30 mg/kg via oral, atipamezole enhanced the AUC of diclofenac by 13.1-fold and the Cmax by 5.6-fold. Similar enhancement also achieved for ABT (100 mg/kg) with AUC and Cmax increased 9.5 and 4.8-fold. As a reversible pan-CYP inhibitor, atipamezole showed less species difference than ABT. It provides a better and easier to use alternative to ABT for ADME optimization and elucidating mechanistic drug metabolism or toxicity studies.

Bacterial steroid hydroxylases: enzyme classes, their functions and comparison of their catalytic mechanisms.

The steroid superfamily includes a wide range of compounds that are essential for living organisms of the animal and plant kingdoms. Structural modifications of steroids highly affect their biological activity. In this review, we focus on hydroxylation of steroids by bacterial hydroxylases, which take part in steroid catabolic pathways and play an important role in steroid degradation. We compare three distinct classes of metalloenzymes responsible for aerobic or anaerobic hydroxylation of steroids, namely: cytochrome P450, Rieske-type monooxygenase 3-ketosteroid 9α-hydroxylase, and molybdenum-containing steroid C25 dehydrogenases. We analyze the available literature data on reactivity, regioselectivity, and potential application of these enzymes in organic synthesis of hydroxysteroids. Moreover, we describe mechanistic hypotheses proposed for all three classes of enzymes along with experimental and theoretical evidences, which have provided grounds for their formulation. In case of the 3-ketosteroid 9α-hydroxylase, such a mechanistic hypothesis is formulated for the first time in the literature based on studies conducted for other Rieske monooxygenases. Finally, we provide comparative analysis of similarities and differences in the reaction mechanisms utilized by bacterial steroid hydroxylases.

Structural characterization of a non-heme iron active site in zeolites that hydroxylates methane.

Iron-containing zeolites exhibit unprecedented reactivity in the low-temperature hydroxylation of methane to form methanol. Reactivity occurs at a mononuclear ferrous active site, α-Fe(II), that is activated by N2O to form the reactive intermediate α-O. This has been defined as an Fe(IV)=O species. Using nuclear resonance vibrational spectroscopy coupled to X-ray absorption spectroscopy, we probe the bonding interaction between the iron center, its zeolite lattice-derived ligands, and the reactive oxygen. α-O is found to contain an unusually strong Fe(IV)=O bond resulting from a constrained coordination geometry enforced by the zeolite lattice. Density functional theory calculations clarify how the experimentally determined geometric structure of the active site leads to an electronic structure that is highly activated to perform H-atom abstraction.

Regulation of Hypoxia-Inducible Factor 1α during Hypoxia by DAP5-Induced Translation of PHD2.

Death-associated protein 5 (DAP5) is an atypical isoform of the translation initiation scaffolds eukaryotic initiation factor 4GI (eIF4GI) and eIF4GII (eIF4GI/II), which recruit mRNAs to ribosomes in mammals. Unlike eIF4GI/II, DAP5 binds eIF2ß, a subunit of the eIF2 complex that delivers methionyl-tRNA to ribosomes. We discovered that DAP5:eIF2ß binding depends on specific stimuli, e.g., protein kinase C (PKC)-Raf-extracellular signal-regulated kinase 1/2 (ERK1/2) signals, and determines DAP5's influence on global and template-specific translation. DAP5 depletion caused an unanticipated surge of hypoxia-inducible factor 1α (HIF-1α), the transcription factor and master switch of the hypoxia response. Physiologically, the hypoxia response is tempered through HIF-1α hydroxylation by the oxygen-sensing prolyl hydroxylase-domain protein 2 (PHD2) and subsequent ubiquitination and degradation. We found that DAP5 regulates HIF-1α abundance through DAP5:eIF2ß-dependent translation of PHD2. DAP5:eIF2-induced PHD2 translation occurred during hypoxia-associated protein synthesis repression, indicating a role as a safeguard to reverse HIF-1α accumulation and curb the hypoxic response.

Probing the role of ceramide hydroxylation in skin barrier lipid models by 2H solid-state NMR spectroscopy and X-ray powder diffraction.

In this work, we studied model stratum corneum lipid mixtures composed of the hydroxylated skin ceramides N-lignoceroyl 6-hydroxysphingosine (Cer[NH]) and α-hydroxylignoceroyl phytosphingosine (Cer[AP]). Two model skin lipid mixtures of the composition Cer[NH] or Cer[AP], N-lignoceroyl sphingosine (Cer[NS]), lignoceric acid (C24:0) and cholesterol in a 0.5:0.5:1:1 molar ratio were compared. Model membranes were investigated by differential scanning calorimetry and 2H solid-state NMR spectroscopy at temperatures from 25 °C to 80 °C. Each component of the model mixture was specifically deuterated for selective detection by 2H NMR. Thus, the exact phase composition of the mixture at varying temperatures could be quantified. Moreover, using X-ray powder diffraction we investigated the lamellar phase formation. From the solid-state NMR and DSC studies, we found that both hydroxylated Cer[NH] and Cer[AP] exhibit a similar phase behavior. At physiological skin temperature of 32 °C, the lipids form a crystalline (orthorhombic) phase. With increasing temperature, most of the lipids become fluid and form a liquid-crystalline phase, which converts to the isotropic phase at higher temperatures (65-80 °C). Interestingly, lignoceric acid in the Cer[NH]-containing mixture has a tendency to form two types of fluid phases at 65 °C. This tendency was also observed in Cer[AP]-containing membranes at 80 °C. While Cer[AP]-containing lipid models formed a short periodicity phase featuring a repeat spacing of d = 5.4 nm, in the Cer[NH]-based model skin lipid membranes, the formation of unusual long periodicity phase with a repeat spacing of d = 10.7 nm was observed.

Biosynthesis and Identification of Metabolites of Maraviroc and Their Use in Experiments to Delineate the Relative Contributions of Cytochrome P4503A4 versus 3A5.

Maraviroc (MVC) is a CCR5 coreceptor antagonist indicated in combination with other antiretroviral agents for the treatment of CCR5-tropic human immunodefinciency virus-1 infection. In this study, the metabolism of MVC was investigated in human liver microsomes to delineate the relative roles of CYP3A4 and CYP3A5. MVC is metabolized to five hydroxylated metabolites, all of which were biosynthesized and identified using mass and NMR spectroscopy. The sites of metabolism were the 2- and 3-positions of the 4,4-difluorocyclohexyl moiety and the methyl of the triazole moiety. Absolute configurations were ultimately ascertained by comparison to authentic standards. The biosynthesized metabolites were used for quantitative in vitro experiments in liver microsomes using cyp3cide, a selective inactivator of CYP3A4. (1S,2S)-2-OH-MVC was the main metabolite representing approximately half of the total metabolism, and CYP3A5 contributed approximately 40% to that pathway in microsomes from CYP3A5*1/*1 donors. The other four metabolites were almost exclusively metabolized by CYP3A4. (1S,2S)-2-hydroxylation also correlated to T-5 N-oxidation, a CYP3A5-specific activity. These data are consistent with clinical pharmacokinetic data wherein CYP3A5 extensive metabolizer subjects showed a modestly lower exposure to MVC.

Photo(geno)toxicity changes associated with hydroxylation of the aromatic chromophores during diclofenac metabolism.

Diclofenac (DCF) can cause adverse reactions such as gastrointestinal, renal and cardiovascular disorders; therefore, topical administration may be an attractive alternative to the management of local pain in order to avoid these side effects. However, previous studies have shown that DCF, in combination with sunlight, displays capability to induce photosensitivity disorders. In humans, DCF is biotransformed into hydroxylated metabolites at positions 4' and 5 (4'OH-DCF and 5OH-DCF), and this chemical change produces non negligible alterations of the drug chromophore, resulting in a significant modification of its light-absorbing properties. In the present work, 5OH-DCF exhibited higher photo(geno)toxic potential than the parent drug, as shown by several in vitro assays (3T3 NRU phototoxicity, DNA ssb gel electrophoresis and COMET), whereas 4'OH-DCF did not display significant photo(geno)toxicity. This could be associated, at least partially with their more efficient UV-light absorption by 5OH-DCF metabolite and with a higher photoreactivity. Interestingly, most of the cellular DNA damage photosensitized by DCF and 5OH-DCF was repaired by the cells after several hours, although this effect was not complete in the case of 5OH-DCF.

Polymorphic Human Sulfotransferase 2A1 Mediates the Formation of 25-Hydroxyvitamin D3-3-O-Sulfate, a Major Circulating Vitamin D Metabolite in Humans.

Metabolism of 25-hydroxyvitamin D3 (25OHD3) plays a central role in regulating the biologic effects of vitamin D in the body. Although cytochrome P450-dependent hydroxylation of 25OHD3 has been extensively investigated, limited information is available on the conjugation of 25OHD3 In this study, we report that 25OHD3 is selectively conjugated to 25OHD3-3-O-sulfate by human sulfotransferase 2A1 (SULT2A1) and that the liver is a primary site of metabolite formation. At a low (50 nM) concentration of 25OHD3, 25OHD3-3-O-sulfate was the most abundant metabolite, with an intrinsic clearance approximately 8-fold higher than the next most efficient metabolic route. In addition, 25OHD3 sulfonation was not inducible by the potent human pregnane X receptor agonist, rifampicin. The 25OHD3 sulfonation rates in a bank of 258 different human liver cytosols were highly variable but correlated with the rates of dehydroepiandrosterone sulfonation. Further analysis revealed a significant association between a common single nucleotide variant within intron 1 of SULT2A1 (rs296361; minor allele frequency = 15% in whites) and liver cytosolic SULT2A1 content as well as 25OHD3-3-O-sulfate formation rate, suggesting that variation in the SULT2A1 gene contributes importantly to interindividual differences in vitamin D homeostasis. Finally, 25OHD3-3-O-sulfate exhibited high affinity for the vitamin D binding protein and was detectable in human plasma and bile but not in urine samples. Thus, circulating concentrations of 25OHD3-3-O-sulfate appear to be protected from rapid renal elimination, raising the possibility that the sulfate metabolite may serve as a reservoir of 25OHD3 in vivo, and contribute indirectly to the biologic effects of vitamin D.

Metabolic Profiling of the Novel Hypoxia-Inducible Factor 2α Inhibitor PT2385 In Vivo and In Vitro.

PT2385 is a first-in-class, selective small-molecule inhibitor of hypoxia-inducible factor-2α (HIF-2α) developed for the treatment of advanced clear cell renal cell carcinoma. Preclinical results demonstrated that PT2385 has potent antitumor efficacy in mouse xenograft models of kidney cancer. It also has activity toward metabolic disease in a mouse model. However, no metabolism data are currently publically available. It is of great importance to characterize the metabolism of PT2385 and identify its effect on systemic homeostasis in mice. High-resolution mass spectrometry-based metabolomics was performed to profile the biotransformation of PT2385 and PT2385-induced changes in endogenous metabolites. Liver microsomes and recombinant drug-metabolizing enzymes were used to determine the mechanism of PT2385 metabolism. Real-time polymerase chain reaction analysis was employed to investigate the reason for the PT2385-induced bile acid dysregulation. A total of 12 metabolites of PT2385 was characterized, generated from hydroxylation (M1, M2), dihydroxylation and desaturation (M3, M4), oxidative-defluorination (M7), glucuronidation (M8), N-acetylcysteine conjugation (M9), and secondary methylation (M5, M6) and glucuronidation (M10, M11, and M12). CYP2C19 was the major contributor to the formation of M1, M2, and M7, UGT2B17 to M8, and UGT1A1/3 to M10-M12. The bile acid metabolites taurocholic acid and tauro-ß-muricholic acid were elevated in serum and liver of mice after PT2385 treatment. Gene expression analysis further revealed that intestinal HIF-2α inhibition by PT2385 treatment upregulated the hepatic expression of CYP7A1, the rate-limiting enzyme in bile acid synthesis. This study provides metabolic data and an important reference basis for the safety evaluation and rational clinical application of PT2385.

Identification of ω- or (ω-1)-Hydroxylated Medium-Chain Acylcarnitines as Novel Urinary Biomarkers for CYP3A Activity.

Quantitative models of endogenous metabolites are useful in predicting CYP3A-mediated drug-drug interactions. This study aimed to identify novel predictive markers for the magnitude of CYP3A induction and inhibition in male and female subjects using an untargeted metabolomics approach. Here we report five ω- or (ω-1)-hydroxylated medium-chain acylcarnitines as novel CYP3A4 markers. As CYP4 catalyzes the ω- or (ω-1)-hydroxylation of various medium-chain fatty acids (MCFAs), recombinant enzyme assays were used to determine the ω- and (ω-1)-hydroxylation activities of CYP3A4, CYP4A11, and CYP4F2. CYP3A4 catalyzed ω- and (ω-1)-hydroxylated MCFAs with the lowest Km and highest Vmax /Km values. Finally, we derived a model to predict midazolam clearance using these markers and demonstrated that the predictive model including three ω- or (ω-1)-hydroxylated medium-chain acylcarnitines, 6ß-OH cortisol, and gender as covariates shows reliable predictability (r2 = 0.894).