Protein and nonprotein cysteinyl thiol modification by N-acetyl-p-benzoquinone imine via a novel ipso adduct.

N-acetyl-p-benzoquinone imine (NAPQI), a reactive metabolite of acetaminophen (APAP), can arylate and oxidize protein and nonprotein thiols in the pathogenesis of APAP-induced hepatotoxicity. We report the first direct evidence for the formation of a labile ipso adduct between glutathione (GSH) and NAPQI using a combination of techniques including liquid chromatography/tandem mass spectrometry and liquid chromatography/NMR spectroscopy. Decomposition kinetics of the GSH-NAPQI ipso adduct and product ratios suggested that the ipso adduct was readily reversible back to NAPQI under neutral and basic conditions. The significance of the ipso adduct is that it may migrate from its site of formation to other cell compartments where it can either oxidize protein thiols or covalently modify them. Ipso adduct formation with protein thiols was demonstrated with a cysteine protease, papain, whose catalytic activity relies on the presence of an active site cysteinyl thiol. The formation and reactions of cysteinyl thiol ipso adducts of NAPQI provides significant new insights into possible reactions of quinone imines with cellular peptides and proteins.     

Rapid oxidation of ring methyl groups is the primary mechanism of biotransformation of gemfibrozil by the fungus <Emphasis Type="Italic">Cunninghamella elegans</Emphasis>

The hypolipidemic agent gemfibrozil (GEM), which has been studied for its metabolism in humans and animals, was investigated to elucidate its primary metabolism by Cunninghamella elegans. The fungus produced ten metabolites (FM1–FM9 and FM6′) from the biotransformation of GEM. Based on LC/MS/MS and NMR analyses, a major metabolite, FM7, was identified as 2′-hydroxymethyl GEM. FM6 was considered to be 5′-hydroxymethyl GEM, after comparison of results LC/MS, LC/MS/MS, and UV absorption spectra to FM7. The combined concentration of FM6 and FM7 was found to increase up to 0.83 mM by day 2, and then decreased gradually with incubation time, followed by a noticeable increase in the biotransformation product, FM1, up to 0.86 mM by day 15. NMR analyses confirmed that FM1 was 2′,5′-dihydroxymethyl GEM. Further minor oxidations of the aromatic ring and carboxylic acid intermediates were also detected. Based upon these findings, the major fungal metabolic pathway for GEM is likely to occur via production of 2′,5′-dihydroxymethyl GEM from 2′-hydroxymethyl GEM. These relatively rapid and diverse biotransformations of GEM by C. elegans suggest that depending upon conditions, it may also follow a similar biodegradation fate when released into the natural environment.     

Adducts between nucleophilic amino acids and hexahydrophthalic anhydride, a structure inducing both types I and IV allergy.

Haptens causing type I allergy have been shown to predominantly form lysine adducts in the carrier protein, while many haptens giving rise to type IV allergy preferentially form adducts with cysteine residues. Hexahydrophthalic anhydride derivatives are strong sensitizers capable of inducing allergic rhinitis, asthma and urticaria (type I allergy) and allergic contact dermatitis (type IV allergy). The ability of hexahydrophthalic anhydride (HHPA) to form adducts with nucleophilic amino acids and a model peptide in vitro is presented. Adduct formation was monitored by high-performance liquid chromatography with ultraviolet light/vis detection (LC-UV/vis) and high-performance liquid chromatography with mass spectrometric detection (LC/MS). The characterization was obtained by nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS and MS/MS). It was found that HHPA formed adducts with N(alpha)-acetylated lysine and cysteine and the non-acetylated alpha-amino group of proline and, to some extent, also with other nucleophilic amino acids. The adducts with lysine and proline were chemically stable. Addition of one HHPA to a model carrier peptide with all important nucleophilic amino acid residues showed N-terminal proline to be the major site of reaction. The addition of a second hapten gave a lysine adduct, but a minor cysteine adduct was also found. The cysteine-HHPA adducts were shown to be chemically unstable and participated in further reactions with lysine forming lysine-HHPA adducts. The results will be useful for understanding the formation of HHPA-protein adducts with the capability of being markers of exposure, and also to a deeper understanding of the chemical structures causing types I and IV allergy.     

S-(N-methylcarbamoyl)glutathione: a reactive S-linked metabolite of methyl isocyanate

S-(N-methylcarbamoyl)glutathione, a chemically-reactive glutathione conjugate, has been isolated from the bile of rats administered methyl isocyanate and characterized, as its N-benzyloxycarbonyl dimethylester derivative, by tandem mass spectrometry. The ability of this glutathione adduct to donate an N-methylcarbamoyl moiety to the free -SH group of cysteine was evaluated in vitro with the aid of a highly specific thermospray LC/MS assay procedure. The glutathione adduct reacted readily with cysteine in buffered aqueous media (pH 7.4, 37 degrees C) and after 2 hr, 42.5% of the substrate existed in the form of S-(N-methylcarbamoyl)cysteine. The reverse reaction, i.e. between the cysteine adduct and free glutathione, also took place readily under these conditions. It is concluded that conjugation of methyl isocyanate with glutathione in vivo affords a reactive S-linked product which displays the potential to carbamoylate nucleophilic amino acids. The various systemic toxicities associated with exposure of animals or humans to methyl isocyanate could therefore be due to release of the isocyanate from its glutathione conjugate, which thus may serve as a vehicle for the transport of methyl isocyanate in vivo.     

The toxicity of opiates and their metabolites in HepG2 cells

The toxicity of codeine (C), codeinone (CO), morphine (M), oxycodone (OC), pholcodine (P) and pholcodine-N-oxide (P-NOX) was assessed in HepG2 cells by determining cell viability via the measurement of lactate dehydrogenase (LDH) leakage through the membrane, depletion of reduced glutathione (GSH) and measurement of total protein content. Incubation of C, M, OC, P or P-NOX with HepG2 cells resulted in no significant loss of cell viability, depletion of GSH or decreased total protein content. In contrast, with CO there was a marked depletion of GSH with significant differences from control cells (P<0.05) being detected after as little as 5 min. This effect preceded the loss of cell viability and the decrease in total protein content. To identify the cause of GSH depletion during incubations with CO, the incubation solutions were analysed by liquid chromatography/tandem mass spectrometry (LC/MS/MS). Analysis showed that a codeinone-glutathione conjugate (CO-SG) had been formed. This adduct was synthesised and characterised by LC/MS/MS and by nuclear magnetic resonance spectroscopy (NMR). CO-SG was quantified in the incubation solutions using the synthesised standard substance. Results obtained in this study support the hypothesis that the toxicity of CO may be partly due to GSH depletion. The absence of LDH leakage and GSH depletion in the incubations containing C or OC suggests, that the presence of both a double bond at Delta 7 and an adjoining keto-group in the 6-position are necessary to elicit the toxicity of M analogues with regard to GSH depletion.     

Global urinary metabolic profiling procedures using gas chromatography-mass spectrometry

The role of urinary metabolic profiling in systems biology research is expanding. This is because of the use of this technology for clinical diagnostic and mechanistic studies and for the development of new personalized health care and molecular epidemiology (population) studies. The methodologies commonly used for metabolic profiling are NMR spectroscopy, liquid chromatography mass spectrometry (LC/MS) and gas chromatography-mass spectrometry (GC/MS). In this protocol, we describe urine collection and storage, GC/MS and data preprocessing methods, chemometric data analysis and urinary marker metabolite identification. Results obtained using GC/MS are complementary to NMR and LC/MS. Sample preparation for GC/MS analysis involves the depletion of urea via treatment with urease, protein precipitation with methanol, and trimethylsilyl derivatization. The protocol described here facilitates the metabolic profiling of ～400-600 metabolites in 120 urine samples per week.     

Adducts between nucleophilic amino acids and hexahydrophthalic anhydride,a structure inducing both types I and IV allergy

Haptens causing type I allergy have been shown to predominantly form lysine adducts in the carrier protein, while many haptens giving rise to type IV allergy preferentially form adducts with cysteine residues. Hexahydrophthalic anhydride derivatives are strong sensitizers capable of inducing allergic rhinitis, asthma and urticaria (type I allergy) and allergic contact dermatitis (type IV allergy). The ability of hexahydrophthalic anhydride (HHPA) to form adducts with nucleophilic amino acids and a model peptide in vitro is presented. Adduct formation was monitored by high-performance liquid chromatography with ultraviolet light/vis detection (LC-UV/vis) and high-performance liquid chromatography with mass spectrometric detection (LC/MS). The characterization was obtained by nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS and MS/MS). It was found that HHPA formed adducts with N^α-acetylated lysine and cysteine and the non-acetylated α-amino group of proline and, to some extent, also with other nucleophilic amino acids. The adducts with lysine and proline were chemically stable. Addition of one HHPA to a model carrier peptide with all important nucleophilic amino acid residues showed N-terminal proline to be the major site of reaction. The addition of a second hapten gave a lysine adduct, but a minor cysteine adduct was also found. The cysteine–HHPA adducts were shown to be chemically unstable and participated in further reactions with lysine forming lysine–HHPA adducts. The results will be useful for understanding the formation of HHPA–protein adducts with the capability of being markers of exposure, and also to a deeper understanding of the chemical structures causing types I and IV allergy.     

A novel protocol to identify and quantify all spin trapped free radicals from in vitro/in vivo interaction of HO(.-) and DMSO: LC/ESR, LC/MS, and dual spin trapping combinations

When dimethyl sulfoxide (DMSO) is oxidized via hydroxyl radical (HO(.-)), it forms methyl radicals ((.-)CH(3)) that can be spin trapped and detected by electron spin resonance (ESR). This ESR spin trapping technique has been widely used in many biological systems to indicate in vivo HO(.-) formation. However, we recently reported that (.-)CH(3) might not be the only carbon-centered radical that was trapped and detected by ESR from in vivo DMSO oxidation. In the present study, newly developed combination techniques consisting of dual spin trapping (free radicals trapped by both regular and deuterated alpha-[4-pyridyl 1]-N-tert-butyl nitrone, d(0)/d(9)-POBN) followed by LC/ESR and LC/MS were used to characterize and quantify all POBN-trapped free radicals from the interaction of HO(.-) and DMSO. In addition to identifying the two well-known free radicals, (.-)CH(3) and (.-)OCH(3), from this interaction, we also characterized two additional free radicals, (.-)CH(2)OH and (.-)CH(2)S(O)CH(3). Unlike ESR, which can measure POBN adducts only in their radical forms, LC/MS identified and quantified all three redox forms, including the ESR-active radical adduct and two ESR-silent forms, the nitrone adduct (oxidized adduct) and the hydroxylamine (reduced adduct). In the bile of rats treated with DMSO and POBN, the ESR-active form of POBN/(.-)CH(3) was not detected. However, with the addition of the LC/MS technique, we found approximately 0.75 microM POBN/(.-)CH(3) hydroxylamine, which represents a great improvement in radical detection sensitivity and reliability. This novel protocol provides a comprehensive way to characterize and quantify in vitro and in vivo free radical formation and will have many applications in biological research.     

Structural characterization of novel inositol phosphosphingolipids of Tritrichomonas foetus and Trichomonas vaginalis

Two major ethanolamine phosphate-substituted inositol phosphosphingolipidshave been identified in the unsaponifiable acidic lipid fractionsof Tritrichomonas foetus and Trichomonas vaginalis. The compoundswere radiolabelled and purified by high-performance thin-layerchromatography followed by high-performance liquid chromatography.The structures were determined by a combination of tandem massspectrometry (MS/MS) and nuclear magnetic resonance (NMR) experiments,and gas—liquid chromatography of components obtained bydegradation and derivatization. Inositol in the T.foetus componentwas 1-linked to the phosphosphingolipid, had the phospho-ethanolaminegroup at the 3-position and a fucosyl residue at the 4-position.The T.vaginalis component lacked the fucosyl moiety. Both organismsalso produced inositol phosphosphingolipids having the samelong-chain base (sphingosine or dihydrosphingosine) and thesame fatty acyl distribution as the inositol diphosphate compounds.These glycosphingolipids may represent metabolic intermediatesfor new types of membrane anchors for surface glycopeptidesor glycolipids that mediate the host—parasite relationshipof these trichomonads. The MS/MS and NMR spectroscopic datashould provide reference information for structural determinationsof other phosphorylated inositol derivatives. inositol phosphosphingolipids NMR tandem MS T.foetus T. vaginalis     

Peroxidatic cysteine residue of peroxiredoxin 2 separated from human red blood cells treated by <Emphasis Type="Italic">tert</Emphasis>-butyl hydroperoxide is hyperoxidized into sulfinic and sulfonic acids

Peroxiredoxin 2 (Prx2) is a redox enzyme that is abundantly expressed in red blood cells (RBCs) and has been the focus of clinical attention for monitoring the oxidative status. We previously developed a method to quantify the reduced and hyperoxidized forms of Prx2 in human RBCs using reverse-phase high-performance liquid chromatography (HPLC). In the present study, we investigated the hyperoxidative status of Prx2 at the molecular level in a post-translational modification analysis using a liquid chromatography–tandem mass spectrometry (LC–MS/MS) system. The LC–MS/MS analysis of the trypsin digests of Prx2 fractionated by reverse-phase HPLC demonstrated that the cysteine-51 residue (Cys-51) of the protein was modified with the hyperoxidative functional groups, sulfinic acid (–SO₂H) and sulfonic acid (–SO₃H), in RBCs treated with tert-butyl hydroperoxide (t-BHP). Furthermore, a selected ion monitoring (SIM) analysis quantitatively showed that sulfinic acid- and sulfonic acid-induced modifications in Prx2 Cys-51 were increased by the treatment with the oxidant. It was demonstrated that the peroxidatic cysteine of Prx2 separated using our HPLC system for oxidative monitoring was hyperoxidized into sulfinic acid and sulfonic acid in RBCs under an oxidative stress condition.     

Use of synthetic oligoribonucleotides to probe RNA-protein interactions in the MS2 translational operator complex.

Synthetic oligoribonucleotides have been used to probe the interaction of MS2 coat protein with the translational operator of the MS2 replicase gene. We have investigated the possible formation of a transient covalent bond between the single-stranded uridine residue, at position -5, and a cysteine side-chain on the coat protein, by the incorporation of a chemically modified residue (5-BrU) at this position. This chemically synthesised operator variant has a binding constant of between 10 and 50 times greater than that of the wild type and is therefore comparable with the tight binding variant having a cytidine substituted at the -5 position. Dissociation kinetics show that the complex with the 5-BrU operator is more stable than the -5C variant; a result which is consistent with the formation of a Michael adduct at the -5 position. In addition, a number of other chemical variants of the operator have been analysed. These include operators incorporating deoxyadenine residues at each of the important single-stranded adenine sites. Recently the Michael adduct proposal has been challenged on the basis of mutagenesis of the coat protein cysteine residues. These results are discussed in the light of our data in support of Michael adduct formation.     

Production of a novel tryptophan analog, beta-1-indazole-L-alanine with tryptophan synthase of Escherichia coli

The tryptophan synthase alpha 2 beta 2 complex from Escherichia coli has been found to catalyze the beta-replacement reaction of L-serine with indazole, an indole analog which has a nitrogen atom at the 2-position (pyrazole ring). The reaction product was isolated and identified as beta-indazolealanine by mass spectrometric, elemental and NMR analyses. Careful assignment of 1H- and 13C-signals with several NMR techniques revealed that the beta-carbon of the product alanine moiety was bound to the 1-N-position of the indazole ring. This is the first example of the beta-replacement reaction catalyzed by tryptophan synthase occurring at any other position than the 3-position of indole analogs.     

Probing the geometric and electronic structures of the low-temperature azide adduct and the product-inhibited form of oxidized manganese superoxide dismutase

The geometric and electronic structures of the six-coordinate azide adduct of oxidized manganese superoxide dismutase (Mn3+ SOD) that is formed at low temperatures, LT N3-Mn3+ SOD, has been examined in detail through a combined spectroscopic/computational approach. Electronic absorption, circular dichroism (CD), magnetic CD (MCD) and variable-temperature, variable-field (VTVH) MCD spectroscopies were used to determine electronic transition energies and to obtain an estimate of zero-field splitting parameters for LT N3-Mn3+ SOD. These experimental data were utilized in conjunction with semiempirical intermediate neglect of differential overlap/spectroscopic parametrization-configuration interaction (INDO/S-CI) and time-dependent density functional theory (TD-DFT) computations to evaluate hypothetical active-site models of LT N3-Mn3+ SOD generated by constrained DFT geometry optimizations. Collectively, our spectroscopic/computational results indicate that N3- binding to Mn3+ SOD at low temperatures promotes neither protonation of the axial solvent ligand nor reorientation of the redox-active molecular orbital, both of which had been previously suggested. Using the same experimentally validated computational approach, models of the product-inhibited form of MnSOD were also developed and evaluated by their relative energies and TD-DFT-computed absorption spectra. On the basis of our computational results as well as previously published kinetic data, we propose that the product-inhibited form of MnSOD is best described as a side-on peroxo-Mn3+ adduct possessing an axial H2O ligand. Notably, attempts to generate a stable hydroperoxo-Mn3+ SOD species by protonation of the proximal O atom of the hydroperoxo ligand resulted in dissociation of HOO- and eventual H+ transfer from Tyr34 to HOO-, generating deprotonated Tyr34 and H2O2. The implications of these results with respect to the mechanism of O2*- dismutation by MnSOD are discussed.     

In-source formation of N-acetyl-p-benzoquinone imine (NAPQI), the putatively toxic acetaminophen (paracetamol) metabolite, after derivatization with pentafluorobenzyl bromide and GC-ECNICI-MS analysis

Pentafluorobenzyl (PFB) bromide (PFB-Br) is a versatile derivatization reagent for numerous classes of compounds. Under electron-capture negative-ion chemical ionization (ECNICI) conditions PFB derivatives of acidic compounds readily and abundantly ionize to produce intense anions due to [M-PFB](-). In the present article we investigated the PFB-Br derivatization of unlabelled acetaminophen (N-acetyl-p-aminophenol, NAPAP-d(0); paracetamol; MW 151) and tetradeuterated acetaminophen (NAPAP-d(4); MW 155) in anhydrous acetonitrile and their GC-ECNICI-MS behavior using methane as the buffer gas. In addition to the expected anions [M-PFB](-) at m/z 150 from NAPAP-d(0) and m/z 154 from NAPAP-d(4), we observed highly reproducibly almost equally intense anions at m/z 149 and m/z 153, respectively. Selected ion monitoring of these ions is suitable for specific and sensitive quantification of acetaminophen in human plasma and urine. Detailed investigations suggest in-source formation of N-acetyl-p-benzoquinone imine (NAPQI; MW 149), the putatively toxic acetaminophen metabolite, from the PFB ether derivative of NAPAP. GC-ECNICI-MS of non-derivatized NAPAP did not produce NAPQI. The peak area ratio of m/z 149 to m/z 150 and of m/z 153 to m/z 154 decreased with increasing ion-source temperature in the range 100-250°C. Most likely, NAPQI formed in the ion-source captures secondary electrons to become negatively charged (i.e., [NAPQI](-)) and thus detectable. Formation of NAPQI was not observed under electron ionization (EI) conditions, i.e., by GC-EI-MS, from derivatized and non-derivatized NAPAP. NAPQI was not detectable in flow injection analysis LC-MS of native NAPAP in positive electrospray ionization (ESI) mode, whereas in negative ESI mode low extent NAPQI formation was observed (<5%). Our results suggest that oxidation of drug derivatives in the ion-sources of mass spectrometers may form intermediates that are produced from activated drugs in enzyme-catalyzed reactions.     

Probing the CYP3A4 active site by cysteine scanning mutagenesis and photoaffinity labeling

The mechanism of CYP3A4-substrate interactions has been investigated using a battery of techniques including cysteine scanning mutagenesis, photoaffinity labeling, and structural modeling. In this study, cysteine scanning mutagenesis was performed at seven sites within CYP3A4 proposed to be involved in substrate interaction and/or cooperativity. Photolabeled CYP3A4 peptide adducts were further characterized by mass spectrometric analysis for each mutant after proteolytic digestion and isolation of fluorescent photolabeled peptides. Among the tryptic peptides of seven tested mutants, three photolabeled peptides of the F108C mutant, ECYSVFTNR (positions 97-105), VLQNFSFKPCK (positions 459-469), and RPCGPVGFMK (positions 106-115) were identified by MALDI-TOF-MS and nano-LC/ESI QTOF MS. The site of modification was further localized to the substituted Cys-108 residue in the mutant peptide adduct RPCGPVGFMK (positions 106-115) by nano-LC/ESI QTOF MS/MS. In summary, we described a potentially useful method to study P450 active sites using a combination of cysteine scanning mutagenesis and photoaffinity labeling.     

Identification of the major constituents of Hypericum perforatum by LC/SPE/NMR and/or LC/MS

The newly established hyphenated instrumentation of LC/DAD/SPE/NMR and LC/UV/(ESI)MS techniques have been applied for separation and structure verification of the major known constituents present in Greek Hypericum perforatum extracts. The chromatographic separation was performed on a C18 column. Acetonitrile-water was used as a mobile phase. For the on-line NMR detection, the analytes eluted from column were trapped one by one onto separate SPE cartridges, and hereafter transported into the NMR flow-cell. LC/DAD/SPE/NMR and LC/UV/MS allowed the characterization of constituents of Greek H. perforatum, mainly naphtodianthrones (hypericin, pseudohypericin, protohypericin, protopseudohypericin), phloroglucinols (hyperforin, adhyperforin), flavonoids (quercetin, quercitrin, isoquercitrin, hyperoside, astilbin, miquelianin, I3,II8-biapigenin) and phenolic acids (chlorogenic acid, 3-O-coumaroylquinic acid). Two phloroglucinols (hyperfirin and adhyperfirin) were detected for the first time, which have been previously reported to be precursors in the biosynthesis of hyperforin and adhyperforin.     

Docking and molecular dynamics studies on triclosan derivatives binding to FabI

FabI, enoyl-ACP reductase (ENR), is the rate-limiting enzyme in the last step for fatty acids biosynthesis in many bacteria. Triclosan (TCL) is a commercial bactericide, and as a FabI inhibitor, it can depress the substrate (trans-2-enoyl-ACP) binding with FabI to hinder the fatty acid synthesis. The structure-activity relationship between TCL derivatives and FabI protein has already been acknowledged, however, their combination at the molecular level has never been investigated. This paper uses the computer-aided approaches, such as molecular docking, molecular dynamics simulation, and binding free energy calculation based on the molecular mechanics/Poisson-Bolzmann surface area (MM/PBSA) method to illustrate the interaction rules of TCL derivatives with FabI and guide the development of new derivatives. The consistent data of the experiment and corresponding activity demonstrates that electron-withdrawing groups on side chain are better than electron-donating groups. 2-Hydroxyl group on A ring, promoting the formation of hydrogen bond, is vital for bactericidal effect; and the substituents at 4-position of A ring, 2′-position and 4′-position of B ring benefit antibacterial activity due to forming a hydrogen bond or stabilizing the conformation of active pocket residues of receptor. While the substituents at 3′-position and 5′-position of B ring destroy the π-π stacking interaction of A ring and NAD⁺ which depresses the antibacterial activity. This study provides a new sight for designing novel TCL derivatives with superior antibacterial activity.     

In vitro metabolic fate of a novel structural class: evidence for the formation of a reactive intermediate on a benzothiophene moiety

The characterization of the metabolic pathways of new chemical entities with a special emphasis on detecting potentially reactive metabolites is increasingly being performed early in the drug discovery process. In the present study, the preliminary in vitro metabolic routes of a series of novel 2-substituted benzothiophene-containing discovery molecules were determined in fresh and cryopreserved hepatocyte suspensions. The objectives of this investigation were: (1) to use systematic LC/MS and LC/MS/MS analyses to provide a preliminary characterization of the in vitro metabolism of these compounds, with a particular focus on metabolites potentially arising from reactive intermediates, and (2) to identify potential lead molecules not associated with such metabolic pathways. This benzothiophene-containing series of compounds was characterized by the formation of five metabolites, at least two of which (dihydrodiol formation and glutathione adduct of the dihydrohydroxyl) were indicative of the formation of a reactive arene oxide intermediate. Tandem mass spectral analysis of the metabolites formed from a variety of structurally similar compounds demonstrated this reactive arene oxide intermediate to form on the 2-substituted benzothiophene moiety. Substitution of the benzothiophene with other functional groups eliminated these potentially toxic metabolites. The data presented here demonstrate the utility of performing metabolic route screens early in the drug discovery process prior to lengthy and costly radiolabeled studies, and furthermore, implicate a 2-substituted benzothiophene moiety as a substrate for formation of a reactive arene oxide intermediate.     

Research strategies for design and development of NSAIDs: clue to balance potency and toxicity of acetanilide compounds

Despite the fact that many modern drug therapies are based on the concept of enzyme inhibition, inhibition of several enzymes leads to pathological disorders. Clinically used nonsteroidal anti-inflammatory drugs (NSAIDs) bind to the active site of the membrane protein, cyclooxygenase (COX) and inhibit the synthesis of prostaglandins, the mediators for causing inflammation. At the same time, inhibition of hepatic cysteine proteases by some NSAID metabolites like NAPQI is implicated in the pathogenesis of hepatotoxicity. As a part of our efforts to develop new effective NSAIDs, a comprehensive investigation starting from synthesis to the study of the final metabolism of acetanilide group of compound has been envisaged with appropriate feedback from kinetic studies to enhance our knowledge and technical competency to feed the know-how to the medicinal chemist to screen out and design new acetanilide derivatives of high potency and low toxicity. Structure-function relationship based on the interaction of acetanilide with its cognate enzyme, cyclooxygenase has been studied critically with adequate comparison with several other available crystal structures of COX-NSAID complexes. Furthermore, to make the receptor based drug design strategy a novel and comprehensive one, both the mechanism of metabolism of acetanilide and structural basis of inhibition of cysteine proteases by the reactive metabolite (NAPQI) formed by cytochrome P450 oxidation of acetanilide have been incorporated in the study. It is hoped that this synergistic approach and the results obtained from such consorted structural investigation at atomic level may guide to dictate synthetic modification with judicious balance between cyclooxygenase inhibition and hepatic cysteine protease inhibition to enhance the potential of such molecular medicine to relieve inflammation on one hand and low hepatic toxicity on the other.     

Conformation and Configuration at the Central Amine Nitrogen of a Nucleotide Adduct of the Carcinogen 2-(Acetylamino)flourene as Studied by 13C and 15N NMR Spectroscopy

Abstract

The conformation and configuration at the central nitrogen of the adduct 8-(N-fluoren-2-ylamino)-2′-deoxyguanosine 5′-monophosphate has been investigated by high-field ¹³C and ¹⁵N NMR spectroscopy. One-bond nitrogen-hydrogen coupling constants and ¹³C chemical shifts for the adduct as well as for the model compounds diphenylamine, 4-nitrodiphenylamine and 2-aminofluorene have been measured in nonaqueous solutions. The data indicate a near planar configuration at the amine nitrogen that links the guanine and fluorene rings of the adduct. The orientations about the guanyl-nitrogen and fluorenyl-nitrogen bonds place the two ring systems in either perpendicular (Type A) or helical (Type B) conformations. It is suggested, based on structural similarities to diarylamines, that the C-N-C bond angle of the adduct is greater than 120° in order to reduce unfavorable steric interactions between the two ring systems. Space-filling molecular models of the adduct in duplex DNA show that the aminofluorene moiety can be oriented into both Type A and Type B conformations within the major groove. The configuration at nitrogen of diphenylamine, 4-nitrodiphenylamine and 2-aminofluorene has also been examined.