Absence Of NADPH Research Articles

L-Glyceraldehyde 3-phosphate reductase from Escherichia coli is a heme binding protein

Recently, we reported that YghZ from Escherichia coli functions as an efficient l-glyceraldehyde 3-phosphate reductase (Gpr). Here we show that Gpr co-purifies with a b-type heme cofactor. Gpr associates with heme in a 1:1 stoichiometry to form a complex that is characterized by a K d value of 5.8 ± 0.2 μM in the absence of NADPH and a K d value of 11 ± 1.3 μM in the presence of saturating NADPH. The absorbance spectrum of reconstituted Gpr indicates that heme is bound in a hexacoordinate low-spin state under both oxidizing and reducing conditions. The physiological function of heme association with Gpr is unclear, as the l-glyceraldehyde 3-phosphate reductase activity of Gpr does not require the presence of the cofactor. Bioinformatics analysis reveals that Gpr clusters with a family of putative monooxygenases in several organisms, suggesting that Gpr may act as a heme-dependent monooxygenase. The discovery that Gpr associates with heme is interesting because Gpr shares 35% amino acid identity with the mammalian voltage-gated K + channel β-subunit, an NADPH-dependent oxidoreductase that endows certain voltage-gated K + channels with hemoprotein-like, O 2-sensing properties. To date the molecular origin of O 2 sensing by voltage-gated K + channels is unknown and the results presented herein suggest a role for heme in this process.

Recombinant Squalene Synthase. Synthesis of Cyclopentyl Non-Head-to-Tail Triterpenes

Incubation of farnesyl diphosphate with recombinant yeast squalene synthase in the absence of NADPH gives a mixture of triterpene hydrocarbons and alcohols, including botryococcene-like compounds with 1'-3 linkages between the farnesyl units. One of these molecules has an unusual cyclopentane structure similar to those recently reported in plant extracts and lakebed sediments.

Characterization of Sulfoxygenation and Structural Implications of Human Flavin-Containing Monooxygenase Isoform 2 (FMO2.1) Variants S195L and N413K

Catalytically active human flavin-containing monooxygenase isoform 2 (FMO2.1) is encoded by an allele detected only in individuals of African or Hispanic origin. Genotyping and haplotyping studies indicate that S195L and N413K occasionally occur secondary to the functional FMO2*1 allele encoding reference protein Gln472. Sulfoxygenation under a range of conditions reveals the role these alterations may play in individuals expressing active FMO2 and provides insight into FMO structure. Expressed S195L lost rather than gained activity as pH was increased or when cholate was present. The activity of S195L was mostly eliminated after heating at 45 degrees C for 5 min in the absence of NADPH, but activity was preserved if NADPH was present. By contrast, Gln472 was less sensitive to heat, a response not affected by NADPH. A major consequence of the S195L mutation was a mean 12-fold increase in K(m) for NADPH compared with Gln472. Modeling an S213L substitution, the equivalent site, in the structural model of FMO from the Methylophaga bacterium leads to disruption of interactions with NADP(+). N413K had the same pattern of activity as Gln472 in response to pH, cholate, and magnesium, but product formation was always elevated by comparison. N413K also lost more activity when heated than Gln472; however, NADPH attenuated this loss. The major effects of N413K were increases in velocity and k(cat) compared with Gln472. Although these allelic variants are expected to occur infrequently as mutations to the FMO2*1 allele, they contribute to our overall understanding of mammalian FMO structure and function.

In Vitro Screening of 50 Highly Prescribed Drugs for Thiol Adduct Formation—Comparison of Potential for Drug-Induced Toxicity and Extent of Adduct Formation

Reactive metabolite formation has been associated with drug-induced liver, skin, and hematopoietic toxicity of many drugs that has resulted in serious clinical toxicity, leading to clinical development failure, black box warnings, or, in some cases, withdrawal from the market. In vitro and in vivo screening for reactive metabolite formation has been proposed and widely adopted in the pharmaceutical industry with the aim of minimizing the property and thus the risk of drug-induced toxicity (DIT). One of the most common screening methods is in vitro thiol trapping of reactive metabolites. Although it is well-documented that many hepatotoxins form thiol adducts, there is no literature describing the adduct formation potential of safer drugs that are widely used. The objective of this study was to quantitatively assess the thiol adduct formation potential of 50 drugs (10 associated with DIT and 40 not associated) and document apparent differences in adduct formation between toxic and safer drugs. Dansyl glutathione was used as a trapping agent to aid the quantitation of adducts following in vitro incubation of drugs with human liver microsomes in the presence and absence of NADPH. Metabolic turnover of these drugs was also monitored by LC/UV. Overall, 15 out of the 50 drugs screened formed detectable levels of thiol adducts. There were general trends toward more positive findings in the DIT group vs the non-DIT group. These trends became more marked when the relative amount of thiol adducts was taken into account and improved further when dose and total daily reactive metabolite burdens were considered. In conclusion, there appears to be a general trend between the extent of thiol adduct formation and the potential for DIT, which would support the preclinical measurement and minimization of the property through screening of thiol adduct formation as part of an overall discovery optimization paradigm.

CYP3A4-Mediated Ester Cleavage as the Major Metabolic Pathway of the Oral Taxane 3′-tert-Butyl-3′-N-tert-butyloxycarbonyl-4-deacetyl-3′-dephenyl-3′-N-debenzoyl-4-O-methoxycarbonyl-paclitaxel (BMS-275183)

3'-tert-Butyl-3'-N-tert-butyloxycarbonyl-4-deacetyl-3'-dephenyl-3'-N-debenzoyl-4-O-methoxycarbonyl-paclitaxel (BMS-275183) is an orally available taxane analog that has the potential to be used as an oral agent to treat cancers. The compound is similar to the two clinically intravenously administered taxanes, paclitaxel and docetaxel, in that it contains a baccatin ring linked to a side chain through an ester bond. Unlike the other taxanes, the hydrolysis of this ester bond leads to formation of a free baccatin core (M13) that was the major metabolism pathway in incubations of [(14)C]BMS-275183 in human liver microsomes (HLMs) in the presence of NADPH, but it was not formed in incubations with human liver cytosol or HLM in the absence of NADPH. The other prominent metabolites formed in HLM incubations resulted from oxidation of t-butyl groups on the side chain (M20, M20B, M21, M22, and M23). All these metabolites were formed by cDNA-expressed CYP3A and not by other cytochrome P450 (P450) enzymes tested. Formation of these metabolites was selectively inhibited by ketoconazole and troleandomycin. The formation of M13 followed Michaelis-Menten kinetics with the K(m) values of 1.3 to 2.4 muM in HLM or CYP3A4; the V(max) value for the formation of M13 and M23 in the cDNA-expressed CYP3A4 matched well (within 2-fold difference) with that determined in HLM when expressed in units of per picomole of P450. These results showed that BMS-275183 is metabolized by CYP3A4 to yield baccatin through oxidation of side-chain t-butyl groups. An intramolecular cyclization of a side-chain hydroxylation metabolite is proposed to be responsible for the formation of M13, the side-chain hydrolysis metabolite.

Abstract 410: Mitochondrial Uncoupling with CCCP Reverses Acute Hyperglycemia Induced Endothelial Dysfunction in Human Arterioles

Background : Acute hyperglycemia (HG) impairs endothelial function, which may be related to hyperpolarization of the inner mitochondrial membrane and excessive production of mitochondrial superoxide (ROS). Mitochondrial uncoupling agents could potentially reverse endothelial dysfunction due to HG in intact human arterioles. Methods : Human adipose arterioles were incubated in either euglycemic (NG, 5mM) or hyperglycemic (HG, 33mM) buffer for 4 hours. Vessels were then exposed to increasing doses of acetylcholine (ACh) in the presence and absence of NADPH oxidase inhibitor gp91ds-tat or CCCP, (100 nM), a mitochondrial membrane uncoupling agent. Vascular superoxide levels were also measured after incubation with NG and HG using a chemiluminescent probe (L-012) in the absence and presence of 1 and 2 above. Results : ACh-induced vasodilation was impaired by HG vs. NG control (P<0.001). This impairment was modestly reversed by gp91 ds-tat (P< 0.001), but endothelial function was completely restored to the level observed under euglycemic conditions by CCCP (P=0.008 vs. HG, P>0.90 vs. NG). Endothelium-dependent vasodilation in HG vessels was greater after CCCP compared to gp91ds-tat (P<0.001). L-NAME inhibited all dilation to ACh indicating endothelium-dependence (data not shown). HG induced an increase in superoxide relative to NG (P<0.001), which was similarly suppressed by gp91ds-tat (P = 0.001) and CCCP (P=0.002). Conclusion : HG-induced endothelial dysfunction is reversed with partial uncoupling of mitochondrial oxidative phosphorylation. Mitochondrial uncoupling more than NADPH oxidase blockade improves endothelial dysfunction due to HG.

In Vitro Metabolism of Pyrethroid Pesticides by Rat and Human Hepatic Microsomes and Cytochrome P450 Isoforms

Species differences in the intrinsic clearance (CL(int)) and the enzymes involved in the metabolism of pyrethroid pesticides were examined in rat and human hepatic microsomes. The pyrethroids bifenthrin, S-bioallethrin, bioresmethrin, beta-cyfluthrin, cypermethrin, cis-permethrin, and trans-permethrin were incubated in rat and human hepatic microsomes in the presence or absence of NADPH. Metabolism was measured using a parent depletion approach. The CL(int) of the pyrethroids was 5- to 15-fold greater in rat relative to human microsomes except for trans-permethrin, which was approximately 45% greater in human microsomes. The metabolism of bifenthrin, S-bioallethrin, and cis-permethrin in rat and human hepatic microsomes was solely the result of oxidative processes. The metabolism of bioresmethrin and cypermethrin in human hepatic microsomes was solely the result of hydrolytic processes. Bioresmethrin and cypermethrin in rat hepatic microsomes and beta-cyfluthrin and trans-permethrin in microsomes from both species were metabolized by both oxidative and hydrolytic pathways. The metabolism of trans-permethrin was reduced when incubated with its diastereomer, cis-permethrin, in both rat and human hepatic microsomes. Rat cytochrome P450 (P450) isoforms that showed activity toward several pyrethroids included CYP1A1, CYP1A2, CYP2C6, CYP2C11, CYP3A1, and CYP3A2. Human P450 isoforms that showed activity toward multiple pyrethroids were CYP2C8, CYP2C9, CYP2C19, and CYP3A4. Species-specific differences in metabolism may result in variable detoxification of pyrethroids, which may in turn result in divergent neurotoxic outcomes. These species differences and isomer interactions in metabolism of pyrethroids should be considered when assessing the potential adverse health effects of pyrethroid pesticides.

Surface plasmon resonance sensor in the analysis of caffeine binding to CYP1A2 p450 monoxygenase in the presence and absence of NADPH

An alternative to liquid chromatography–mass spectroscopic analysis of CYP catalysis of candidate drugs is proposed by a surface plasmon resonance method. By covalently linking microsomes containing human recombinant CYP1A2, NADPH reductase and cytochrome b5 to a dithiodipropionic acid self-assembly monolayer formed on a gold-coated glass slide, a surface plasmon resonance (SPR) waveguide sensor is investigated. Previously only the CYP monoxygenases have been immobilised for SPR experiments, which bring into question the accuracy of association and dissociation constants determined in the absence of the other essential components (NADPH, NADPH reductase, cytochrome b5) for CYP activity. Caffeine was used as a standard substrate of CYP1A2 and comparisons in the presence and the absence of NADPH were performed to investigate the influence of enabling catalysis upon kinetic constants. This has consequences for future CYP bioassay design as an alternative measure for CYP enzyme kinetics which is demonstrated. The effect of concentration on response time and angular change in the plasmon resonances was also observed, with potential applications for determining metabolism of compounds by this method.

Chemical Reactivity of Methoxy 4-O-Aryl Quinolines: Identification of Glutathione Displacement Products in Vitro and in Vivo

AMG 458 {1-(2-hydroxy-2-methylpropyl)-N-[5-(7-methoxyquinolin-4-yloxy)pyridin-2-yl]-5-methyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide} is a potent, selective inhibitor of c-Met, a receptor tyrosine kinase that is often deregulated in cancer. AMG 458 was observed to bind covalently to liver microsomal proteins from rats and humans in the absence of NADPH. When [(14)C]AMG 458 was incubated with liver microsomes in the presence of glutathione and N-acetyl cysteine, thioether adducts were detected by radiochromatography and LC/MS/MS analysis. These adducts were also formed upon incubation of AMG 458 with glutathione and N-acetyl cysteine in buffers at pH 7.4. In vivo, the thioether adducts were detected in bile and urine of bile duct-cannulated rats dosed with [(14)C]AMG 458. The two adducts were isolated, and their structures were determined by MS/MS and NMR analysis. The identified structures resulted from a thiol displacement reaction to yield a quinoline thioether structure and the corresponding hydroxyaryl moiety. The insights gained from elucidating the mechanism of adduct formation led to the design of AMG 458 analogues that exhibited eliminated or reduced glutathione adduct formation in vitro and in vivo.

Can In Vitro Metabolism-Dependent Covalent Binding Data in Liver Microsomes Distinguish Hepatotoxic from Nonhepatotoxic Drugs? An Analysis of 18 Drugs with Consideration of Intrinsic Clearance and Daily Dose

In vitro covalent binding assessments of drugs have been useful in providing retrospective insights into the association between drug metabolism and a resulting toxicological response. On the basis of these studies, it has been advocated that in vitro covalent binding to liver microsomal proteins in the presence and the absence of NADPH be used routinely to screen drug candidates. However, the utility of this approach in predicting toxicities of drug candidates accurately remains an unanswered question. Importantly, the years of research that have been invested in understanding metabolic bioactivation and covalent binding and its potential role in toxicity have focused only on those compounds that demonstrate toxicity. Investigations have not frequently queried whether in vitro covalent binding could be observed with drugs with good safety records. Eighteen drugs (nine hepatotoxins and nine nonhepatotoxins in humans) were assessed for in vitro covalent binding in NADPH-supplemented human liver microsomes. Of the two sets of nine drugs, seven in each set were shown to undergo some degree of covalent binding. Among hepatotoxic drugs, acetaminophen, carbamazepine, diclofenac, indomethacin, nefazodone, sudoxicam, and tienilic acid demonstrated covalent binding, while benoxaprofen and felbamate did not. Of the nonhepatotoxic drugs evaluated, buspirone, diphenhydramine, meloxicam, paroxetine, propranolol, raloxifene, and simvastatin demonstrated covalent binding, while ibuprofen and theophylline did not. A quantitative comparison of covalent binding in vitro intrinsic clearance did not separate the two groups of compounds, and in fact, paroxetine, a nonhepatotoxin, showed the greatest amount of covalent binding in microsomes. Including factors such as the fraction of total metabolism comprised by covalent binding and the total daily dose of each drug improved the discrimination between hepatotoxic and nontoxic drugs based on in vitro covalent binding data; however, the approach still would falsely identify some agents as potentially hepatotoxic.

The Molecular Cloning of Artemisinic Aldehyde Δ11(13) Reductase and Its Role in Glandular Trichome-dependent Biosynthesis of Artemisinin in Artemisia annua

At some point during biosynthesis of the antimalarial artemisinin in glandular trichomes of Artemisia annua, the Delta11(13) double bond originating in amorpha-4,11-diene is reduced. This is thought to occur in artemisinic aldehyde, but other intermediates have been suggested. In an effort to understand double bond reduction in artemisinin biosynthesis, extracts of A. annua flower buds were investigated and found to contain artemisinic aldehyde Delta11(13) double bond reductase activity. Through a combination of partial protein purification, mass spectrometry, and expressed sequence tag analysis, a cDNA clone corresponding to the enzyme was isolated. The corresponding gene Dbr2, encoding a member of the enoate reductase family with similarity to plant 12-oxophytodienoate reductases, was found to be highly expressed in glandular trichomes. Recombinant Dbr2 was subsequently characterized and shown to be relatively specific for artemisinic aldehyde and to have some activity on small alpha,beta-unsaturated carbonyl compounds. Expression in yeast of Dbr2 and genes encoding four other enzymes in the artemisinin pathway resulted in the accumulation of dihydroartemsinic acid. The relevance of Dbr2 to trichome-specific artemisinin biosynthesis is discussed.

BYK191023 (2-[2-(4-Methoxy-pyridin-2-yl)-ethyl]-3H-imidazo[4,5-b]pyridine) Is an NADPH- and Time-Dependent Irreversible Inhibitor of Inducible Nitric-Oxide Synthase

Imidazopyridine derivates were recently shown to be a novel class of selective and arginine-competitive inhibitors of inducible nitric-oxide synthase (iNOS), and 2-[2-(4-methoxypyridin-2-yl)-ethyl]-3H-imidazo[4,5-b]pyridine (BYK191023) was found to have very high selectivity in enzymatic and cellular models ( Mol Pharmacol 69: 328-337, 2006 ). Here, we show that BYK191023 irreversibly inactivates murine iNOS in an NADPH- and time-dependent manner, whereas it acts only as a reversible l-arginine-competitive inhibitor in the absence of NADPH or during anaerobic preincubation. Time-dependent irreversible inhibition by BYK191023 could also be demonstrated in intact cells using the RAW macrophage or iNOS-overexpressing human embryonic kidney 293 cell lines. The mechanism of BYK191023 inhibition in the presence of NADPH was studied using spectral, kinetic, chromatographic, and radioligand binding methods. BYK191023-bound iNOS was spectrally indistinguishable from l-arginine-bound iNOS, pointing to an interaction of BYK191023 with the catalytic center of the enzyme. [(3)H]BYK191023 was recovered quantitatively from irreversibly inactivated iNOS, and no inhibitor metabolite was detected by high-performance liquid chromatography (HPLC). Size exclusion chromatography revealed only about 20% iNOS dissociation into monomers. Furthermore, HPLC and spectrophotometric analysis showed that the irreversible inhibition was associated with loss of heme from iNOS and a reduced ability to form the distinctive ferrous heme-CO complex (cytochrome P450). Thus, enzyme inactivation is mainly caused by heme loss, and it occurs in the inhibitor-bound enzyme in the presence of electron flux from NADPH.

Mechanistic Studies of Bacillus subtilis QueF, the Nitrile Oxidoreductase Involved in Queuosine Biosynthesis

The enzyme QueF was recently identified as an enzyme involved in the biosynthesis of queuosine, a 7-deazaguanosine modified nucleoside found in bacterial and eukaryotic tRNA. QueF exhibits sequence homology to the type I GTP cyclohydrolases characterized by FolE, but contrary to the predictions based on sequence analysis the enzyme in fact catalyzes a mechanistically unrelated reaction, the NADPH-dependent reduction of 7-cyano-7-deazaguanine (preQ0) to 7-aminomethyl-7-deazaguanine (preQ1), a late step in the queuosine pathway. The reduction of a nitrile is unprecedented in biology, and we report here characterization and mechanistic studies of the enzyme from Bacillus subtilis. The recombinant enzyme exhibits optimal activity at pH 7.5 and moderate ionic strength, and is not dependent on metal ions for catalytic activity. Steady-state kinetic analysis provided a kcat = 0.66 +/- 0.04 min-1, KM (preQ0) = 0.237 +/- 0.045 microM, and KM (NADPH) = 19.2 +/- 1.1 microM. Based on sequence analysis and homology modeling we predicted previously that Cys55 would be present in the active site and in proximity to the nitrile group of preQ0. Consistent with that prediction we observed that the enzyme was inactivated when preincubated with iodoacetamide, and protected from inactivation when preQ0 was present. Furthermore, titrations of the enzyme with preQ0 in the absence of NADPH were accompanied by the appearance of a new absorption band at 376 nm in the UV-vis spectrum consistent with the formation of an alpha,beta-unsaturated thioimide. Site-directed mutagenesis of Cys55 to Ala or Ser resulted in loss of catalytic activity and no absorption at 376 nm upon addition of preQ0. Based on our data we propose a chemical mechanism for the enzyme-catalyzed reaction, and a chemical rationale for the observation of covalent catalysis.

Glutamate recognition and hydride transfer by Escherichia coli glutamyl‐tRNA reductase

The initial step of tetrapyrrole biosynthesis in Escherichia coli involves the NADPH-dependent reduction by glutamyl-tRNA reductase (GluTR) of tRNA-bound glutamate to glutamate-1-semialdehyde. We evaluated the contribution of the glutamate moiety of glutamyl-tRNA to substrate specificity in vitro using a range of substrates and enzyme variants. Unexpectedly, we found that tRNA(Glu) mischarged with glutamine was a substrate for purified recombinant GluTR. Similarly unexpectedly, the substitution of amino acid residues involved in glutamate side chain binding (S109A, T49V, R52K) or in stabilizing the arginine 52 glutamate interaction (glutamate 54 and histidine 99) did not abrogate enzyme activity. Replacing glutamine 116 and glutamate 114, involved in glutamate-enzyme interaction near the aminoacyl bond to tRNA(Glu), by leucine and lysine, respectively, however, did abolish reductase activity. We thus propose that the ester bond between glutamate and tRNA(Glu) represents the crucial determinant for substrate recognition by GluTR, whereas the necessity for product release by a 'back door' exit allows for a degree of structural variability in the recognition of the amino acid moiety. Analyzing the esterase activity, which occured in the absence of NADPH, of GluTR variants using the substrate 4-nitrophenyl acetate confirmed the crucial role of cysteine 50 for thioester formation. Finally, the GluTR variant Q116L was observed to lack reductase activity whereas esterase activity was retained. Structure-based molecular modeling indicated that glutamine 116 may be crucial in positioning the nicotinamide group of NADPH to allow for productive hydride transfer to the substrate. Our data thus provide new information about the distinct function of active site residues of GluTR from E. coli.

Functional activity of the mouse flavin‐containing monooxygenase forms 1, 3, and 5

Three functional mouse flavin-containing monooxygenases (mFMOs) (i.e., mFMO1, mFMO3, and mFMO5) have been reported to be the major FMOs present in mouse liver. To examine the biochemical features of these enzymes, recombinant enzymes were expressed as maltose-binding protein fusion proteins (i.e., MBP-mFMO1, MBP-mFMO3, and MBP-mFMO5) in Escherichia coli and isolated and purified with affinity chromatography. The substrate specificity of these three mouse hepatic FMO enzymes were examined using a variety of substrates, including mercaptoimidazole, trimethylamine, S-methyl esonarimod, and an analog thereof, and a series of 10-(N,N-dimethylaminoalkyl)-2-(trifluoromethyl)phenothiazine analogs. The kinetic parameters of the three mouse FMOs for these substrates were compared in an attempt to explore substrate structure--function relationships specific for each mFMO. Utilizing a common phenothiazine substrate for all three enzymes, we compared the pH dependence for the recombinant enzymes under similar conditions. In addition, thermal stability for mFMO1, mFMO3, and mFMO5 enzymes was examined in the presence and absence of NADPH. The results revealed unique features for mFMO5, suggesting possible impact on the functional significance of this abundantly expressed FMO5 isoform in both human and mouse liver.

Species Differences in the in Vitro Metabolism of Deltamethrin and Esfenvalerate: Differential Oxidative and Hydrolytic Metabolism by Humans and Rats

Pyrethroids are neurotoxic pesticides whose pharmacokinetic behavior plays a role in their potency. This study examined the elimination of esfenvalerate and deltamethrin from rat and human liver microsomes. A parent depletion approach in the presence and absence of NADPH was used to assess species differences in biotransformation pathways, rates of elimination, and intrinsic hepatic clearance. Esfenvalerate was eliminated primarily via NADPH-dependent oxidative metabolism in both rat and human liver microsomes. The intrinsic hepatic clearance (CL(INT)) of esfenvalerate was estimated to be 3-fold greater in rodents than in humans on a per kilogram body weight basis. Deltamethrin was also eliminated primarily via NADPH-dependent oxidative metabolism in rat liver microsomes; however, in human liver microsomes, deltamethrin was eliminated almost entirely via NADPH-independent hydrolytic metabolism. The CL(INT) for deltamethrin was estimated to be 2-fold more rapid in humans than in rats on a per kilogram body weight basis. Metabolism by purified rat and human carboxylesterases (CEs) were used to further examine the species differences in hydrolysis of deltamethrin and esfenvalerate. Results of CE metabolism revealed that human carboxylesterase 1 (hCE-1) was markedly more active toward deltamethrin than the class 1 rat CEs hydrolase A and B and the class 2 human CE (hCE-2); however, hydrolase A metabolized esfenvalerate 2-fold faster than hCE-1, whereas hydrolase B and hCE-1 hydrolyzed esfenvalerate at equal rates. These studies demonstrate a significant species difference in the in vitro pathways of biotransformation of deltamethrin in rat and human liver microsomes, which is due in part to differences in the intrinsic activities of rat and human carboxylestersases.

FORMATION OF N-ALKYLPROTOPORPHYRIN IX FROM METABOLISM OF DIALLYL SULFONE IN LUNG AND LIVER

Diallyl sulfone (DASO2) is a garlic derivative formed during cooking or after ingestion. Bioactivation of DASO2 in murine lung and liver results in formation of an epoxide that inactivates CYP2E1 and significantly decreases cytochrome P450 and heme levels. In this study, we tested the hypothesis that DASO2 metabolism leads to production of the heme adduct, N-alkylprotoporphyrin IX (N-alkylPP). Formation of N-alkylPP in vivo and in vitro was determined by spectrophotometric and fluorometric methods, respectively. In in vivo studies, N-alkylPP was generated in the livers of male and female mice treated with DASO2, but was not detectable in the lungs of DASO2-treated mice. In in vitro studies, rates of formation of N-alkylPP in liver and lung microsomes incubated with DASO2 and NADPH were dependent on time and protein concentrations, but were negligible in control incubations performed in the absence of NADPH or DASO2 or with boiled microsomes. The rates of N-alkylPP formation generated in murine liver were higher than those in either murine lung or human liver. Kinetic analysis revealed that murine liver microsomes metabolized DASO2 to N-alkylPP with higher affinity and catalytic efficiency than did murine lung or human liver microsomes. Recombinant rat CYP2E1 also metabolized DASO2 to N-alkylPP; however, rates of formation of the heme adduct was minimal in incubations of recombinant human CYP2E1 with DASO2. These findings demonstrated that the N-alkylPP adduct was produced via metabolism of DASO2 in murine liver and lung microsomes, in human liver microsomes, in recombinant CYP2E1, and in vivo in murine liver.

Non-oxygen-forming pathways of hydrogen peroxide degradation by bovine liver catalase at low hydrogen peroxide fluxes

Heme catalases are considered to degrade two molecules of H2O2 to two molecules of H2O and one molecule of O2 employing the catalatic cycle. We here studied the catalytic behaviour of bovine liver catalase at low fluxes of H2O2 (relative to catalase concentration), adjusted by H2O2-generating systems. At a ratio of a H2O2 flux (given in μM/min− 1) to catalase concentration (given in μM) of 10 min− 1 and above, H2O2 degradation occurred via the catalatic cycle. At lower ratios, however, H2O2 degradation proceeded with increasingly diminished production of O2. At a ratio of 1 min− 1, O2 formation could no longer be observed, although the enzyme still degraded H2O2. These results strongly suggest that at low physiological H2O2 fluxes H2O2 is preferentially metabolised reductively to H2O, without release of O2. The pathways involved in the reductive metabolism of H2O2 are presumably those previously reported as inactivation and reactivation pathways. They start from compound I and are operative at low and high H2O2 fluxes but kinetically outcompete the reaction of compound I with H2O2 at low H2O2 production rates. In the absence of NADPH, the reducing equivalents for the reductive metabolism of H2O2 are most likely provided by the protein moiety of the enzyme. In the presence of NADPH, they are at least in part provided by the coenzyme.

Addressing Metabolic Activation as an Integral Component of Drug Design

Formation of reactive intermediates by metabolism of xenobiotics represents a potential liability in drug discovery and development. Although it is difficult, if not impossible, to predict toxicities of drug candidates accurately, it is prudent to try to minimize bioactivation liabilities as early as possible in the stage of drug discovery and lead optimization. Measurement of covalent binding to liver microsomal proteins in the presence and the absence of NADPH, as well as the use of trapping agents such as glutathione or cyanide ions to provide structural information on reactive intermediates, have been used routinely to screen drug candidates. These in vitro experiments are often supplemented with in vivo covalent binding data in rats. The resulting data are not only used to eliminate potentially risky compounds, but, more importantly, they provide invaluable information to direct the Medicinal Chemistry group efforts to design analogs with less propensity to undergo bioactivation. Select case histories are presented in which this approach was successfully applied at Merck.

Nox4 NAD(P)H Oxidase Mediates Hypertrophy and Fibronectin Expression in the Diabetic Kidney

Renal hypertrophy and extracellular matrix accumulation are early features of diabetic nephropathy. We investigated the role of the NAD(P)H oxidase Nox4 in generation of reactive oxygen species (ROS), hypertrophy, and fibronectin expression in a rat model of type 1 diabetes induced by streptozotocin. Phosphorothioated antisense (AS) or sense oligonucleotides for Nox4 were administered for 2 weeks with an osmotic minipump 72 h after streptozotocin treatment. Nox4 protein expression was increased in diabetic kidney cortex compared with non-diabetic controls and was down-regulated in AS-treated animals. AS oligonucleotides inhibited NADPH-dependent ROS generation in renal cortical and glomerular homogenates. ROS generation by intact isolated glomeruli from diabetic animals was increased compared with glomeruli isolated from AS-treated animals. AS treatment reduced whole kidney and glomerular hypertrophy. Moreover, the increased expression of fibronectin protein was markedly reduced in renal cortex including glomeruli of AS-treated diabetic rats. Akt/protein kinase B and ERK1/2, two protein kinases critical for cell growth and hypertrophy, were activated in diabetes, and AS treatment almost abolished their activation. In cultured mesangial cells, high glucose increased NADPH oxidase activity and fibronectin expression, effects that were prevented in cells transfected with AS oligonucleotides. These data establish a role for Nox4 as the major source of ROS in the kidneys during early stages of diabetes and establish that Nox4-derived ROS mediate renal hypertrophy and increased fibronectin expression.