Side-chain Nitrogen Research Articles

The Singlet Oxygen Oxidation of Chlorpromazine and Some Phenothiazine Derivatives. Products and Reaction Mechanisms

A kinetic and product study of the reactions of chlorpromazine 1, N-methylphenothiazine 2, and N-ethylphenothiazine 3 with singlet oxygen was carried out in MeOH and MeCN. 1 undergoes exclusive side-chain cleavage, whereas the reactions of 2 and 3, in MeOH, afforded only the corresponding sulfoxides. A mechanism for the reaction of 1 is proposed where the first step involves an interaction between singlet oxygen and the side-chain dimethylamino nitrogen. This explains why no side-chain cleavage is observed for 2 and 3.

The Crystal Structure of Cytochrome P460 ofNitrosomonas europaeaReveals a Novel Cytochrome Fold and Heme−Protein Cross-link,

We have determined the 1.8 A X-ray crystal structure of a monoheme c-type cytochrome, cytochrome P460, from Nitrosomonas europea. The chromophore possesses unusual spectral properties analogous to those of the catalytic heme P460 of hydroxylamine oxidoreductase (HAO), the only known heme in biology to withdraw electrons from an iron-coordinated substrate. The analysis reveals a homodimeric structure and elucidates a new c-type cytochrome fold that is predominantly beta-sheet. In addition to the two cysteine thioether links to the porphyrin typical of c-type hemes, there is a third proteinaceous link involving a conserved lysine. The covalent bond is between the lysine side-chain nitrogen and the 13'-meso carbon of the heme, which, following cross-link formation, is sp3-hybridized, demonstrating the loss of conjugation at this position within the porphyrin. The structure has implications for the analogous tyrosine-heme meso carbon cross-link observed in HAO.

Transamidation of Wheat Flour Inhibits the Response to Gliadin of Intestinal T Cells in Celiac Disease

Celiac disease is characterized by activation of HLA-DQ2/DQ8-restricted intestinal gluten-specific CD4(+) T cells. In particular, gluten becomes a better T-cell antigen following deamidation catalyzed by tissue transglutaminase. To date, the only available therapy is represented by adherence to a gluten-free diet. Here, we examined a new enzyme strategy to preventively abolish gluten activity. Enzyme modifications of the immunodominant alpha-gliadin peptide p56-68 were analyzed by mass spectrometry, and peptide binding to HLA-DQ2 was simulated by modeling studies. Wheat flour was treated with microbial transglutaminase and lysine methyl ester; gliadin was subsequently extracted, digested, and deamidated. Gliadin-specific intestinal T-cell lines (iTCLs) were generated from biopsy specimens from 12 adult patients with celiac disease and challenged in vitro with different antigen preparations. Tissue transglutaminase-mediated transamidation with lysine or lysine methyl ester of p56-68 or gliadin in alkaline conditions inhibited the interferon gamma expression in iTCLs; also, binding to DQ2 was reduced but not abolished, as suggested by in silico analysis. Lysine methyl ester was particularly effective in abrogating the activity of gliadin. Notably, a block in the response was observed when iTCLs were challenged with gliadin extracted from flour pretreated with microbial transglutaminase and lysine methyl ester. Transamidation of wheat flour with a food-grade enzyme and an appropriate amine donor can be used to block the T cell-mediated gliadin activity. Considering the crucial role of adaptive immunity in celiac disease, our findings highlight the potential of the proposed treatment to prevent cereal toxicity.

A general approach to the screening and confirmation of tryptamines and phenethylamines by mass spectral fragmentation

Certain characteristic fragmentations of tryptamines (indoleethylamine) and phenethylamines are described. Based on the GC–EI/MS, LC–ESI/MS and MALDI/TOFMS, the mass fragmentations of 13 standard compounds, including α-methyltryptamine (AMT), N, N-dimethyltryptamine (DMT), 5-methoxy-α-methyltryptamine (5-MeO-AMT), N, N-diethyltryptamine (DET), N, N-dipropyltryptamine (DPT), N, N-dibutyltryptamine (DBT), N, N-diisopropyltryptamine (DIPT), 5-methoxy- N, N-dimethyltryptamine (5-MeO-DMT), 5-methoxy- N, N-diisopropyltryptamine (5-MeO-DIPT), methamphetamine (MAMP), 3,4-methylenedioxyamphetamine (3,4-MDA), 3,4-methylenedioxymethamphetamine (3,4-MDMA) and 2-methylamino-1-(3,4-methylenedioxyphenyl)butane (MBDB), were compared. As a result, the parent ions of these analytes were hard to be obtained by GC/MS whereas the protonated molecular ions can be observed clearly by means of ESI/MS and MALDI/TOFMS. Furthermore, two major characteristic fragmentations, namely and α-cleavage ([ M + H] + → [3-vinylindole] +) and β-cleavage ([ M + H] + → [CH 2N +R N1R N2]), are produced when the ESI and MALDI modes are used, respectively. In the case of ESI/MS, the fragment obtained from α-cleavage is the major process. In contrast to this, in the case of MALDI/TOFMS, the major fragment is produced via β-cleavage. The ionization efficiency and fragments formed from either α- or β-cleavages are closely related to the degree of alkylation of the side chain nitrogen in both cases.

The intramolecular δ15N of lysine responds to respiratory status in Paracoccus denitrificans

Presented here is the first experimental evidence that natural, intramolecular, isotope ratios are sensitive to physiological status, based on observations of intramolecular delta(15)N of lysine in the mitochondrial mimic Paracoccus denitrificans. Paracoccus denitrificans, a versatile, gram-negative bacterium, was grown either aerobically or anaerobically on isotopically-characterized ammonium as sole cell-nitrogen source. Nitrogen isotope composition of the biomass with respect to source ammonium was Delta(15)N(cell - NH4) = delta(15) - delta(15)N(NH4) = -6.2 +/- 1.2 per thousand for whole cells under aerobic respiration, whereas cells grown anaerobically produced no net fractionation (Delta(15)N(cell - NH4) = -0.3 +/- 0.23 per thousand). Fractionation of (15)N between protein nitrogen and total cell nitrogen increased during anaerobic respiration and suggests that residual nitrogen-containing compounds in bacterial cell membranes are isotopically lighter under anaerobic respiration. In aerobic cells, the lysine intramolecular difference between peptide and sidechain nitrogen is negligible, but in anaerobic cells was a remarkable Delta(15)N(p - s) = delta(15)N(peptide) - delta(15)N(sidechain) = +11.0 per thousand, driven predominantly by enrichment at the peptide N. Consideration of known lysine pathways suggests this to be likely due to enhanced synthesis of peptidoglycans in the anaerobic state. These data indicate that distinct pathway branching ratios associated with microbial respiration can be detected by natural intramolecular Deltadelta(15)N measurements, and are the first in vivo observations of position-specific measurements of nitrogen isotope fractionation.

Total Synthesis of Flustramine C via Dimethylallyl Rearrangement

The marine natural product flustramine C from the bryozoan Flustra foliacea was synthesized in five steps and 38% yield starting from Nb-methyltryptamine. The key step is the biomimetic oxidation of the natural product deformylflustrabromine causing selective 1,2-rearrangement of the inverse prenyl group. By 1H,15N HMBC experiments, it is unambiguously shown that the reaction with t-BuOCl commences with chlorination of the side chain nitrogen. Deformylflustrabromine itself was synthesized via Danishefsky inverse prenylation. [reaction: see text].

A New Method for the Synthesis of Functionalized Maleimides.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

Crystallographic analysis of calcium-dependent heparin binding to annexin A2.

Annexin A2 and heparin bind to one another with high affinity and in a calcium-dependent manner, an interaction that may play a role in mediating fibrinolysis. In this study, three heparin-derived oligosaccharides of different lengths were co-crystallized with annexin A2 to elucidate the structural basis of the interaction. Crystal structures were obtained at high resolution for uncomplexed annexin A2 and three complexes of heparin oligosaccharides bound to annexin A2. The common heparin-binding site is situated at the convex face of domain IV of annexin A2. At this site, annexin A2 binds up to five sugar residues from the nonreducing end of the oligosaccharide. Unlike most heparin-binding consensus patterns, heparin binding at this site does not rely on arrays of basic residues; instead, main-chain and side-chain nitrogen atoms and two calcium ions play important roles in the binding. Especially significant is a novel calcium-binding site that forms upon heparin binding. Two sugar residues of the heparin derivatives provide oxygen ligands for this calcium ion. Comparison of all four structures shows that heparin binding does not elicit a significant conformational change in annexin A2. Finally, surface plasmon resonance measurements were made for binding interactions between annexin A2 and heparin polysaccharide in solution at pH 7.4 or 5.0. The combined data provide a clear basis for the calcium dependence of heparin binding to annexin A2.

Role of glutamine 148 of human 15-hydroxyprostaglandin dehydrogenase in catalytic oxidation of prostaglandin E2

NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a member of the short-chain dehydrogenase/reductase (SDR) family, catalyzes the first step in the catabolic pathways of prostaglandins and lipoxins. This enzyme oxidizes the C-15 hydroxyl group of prostaglandins and lipoxins to produce 15-keto metabolites which exhibit greatly reduced biological activities. A three-dimensional (3D) structure of 15-PGDH based on the crystal structures of the levodione reductase and tropinone reductase-II was generated and used for docking study with NAD+ coenzyme and PGE2 substrate. Three well-conserved residues among SDR family which correspond to Ser-138, Tyr-151, and Lys-155 of 15-PGDH have been shown to participate in the catalytic reaction. Based on the molecular interactions observed from 3D structure of 15-PGDH, we further propose that Gln-148 in 15-PGDH is important in properly positioning the 15-hydroxyl group of PGE2 by hydrogen bonding with the side-chain oxygen atom of Gln-148. This residue is found to be less conserved and replaceable by glutamyl, histidinyl, and asparaginyl residues in SDR family. Accordingly, site-directed mutagenesis of Gln-148 of 15-PGDH to alanine, glutamic acid, histidine, and asparagine (Q148A, Q148E, Q148H, and Q148N) was carried out. The activity of mutant Q148A was not detectable, whereas those of mutants Q148E, Q148H, and Q148N were comparable to or higher than the wild type. This indicates that the side-chain oxygen or nitrogen atom at position 148 of 15-PGDH plays an important role in anchoring C-15 hydroxyl group of PGE2 through hydrogen bonding for catalytic reaction.

Nickel Superoxide Dismutase Reaction Mechanism Studied by Hybrid Density Functional Methods

The reaction mechanism for the disproportionation of the toxic superoxide radical to molecular oxygen and hydrogen peroxide by the nickel-dependent superoxide dismutase (NiSOD) has been studied using the B3LYP hybrid DFT method. Based on the recent X-ray structures of the enzyme in the resting oxidized Ni(III) and X-ray-reduced Ni(II) states, the model investigated includes the backbone spacer of six residues (sequence numbers 1-6) as a structural framework. The side chains of residues His1, Cys2, and Cys6, which are essential for nickel binding and catalysis, were modeled explicitly. The catalytic cycle consists of two half-reactions, each initiated by the successive substrate approach to the metal center. The two protons necessary for the dismutation are postulated to be delivered concertedly with the superoxide radical anions. The first (reductive) phase involves Ni(III) reduction to Ni(II), and the second (oxidative) phase involves the metal reoxidation back to its resting state. The Cys2 thiolate sulfur serves as a transient protonation site in the interim between the two half-reactions, allowing for the dioxygen and hydrogen peroxide molecules to be released in the reductive and oxidative phases, respectively. The His1 side chain nitrogen and backbone amides of the active site channel are shown to be less favorable transient proton locations, as compared to the Cys2 sulfur. Comparisons are made to the Cu- and Zn-dependent SOD, studied previously using similar models.

A new method for the synthesis of functionalized maleimides

An effective route to novel maleimides is described, which involves the reaction of an enamine derived from the addition of a secondary amine to a dialkyl acetylenedicarboxylate with an arylsulfonyl isocyanate. These maleimides in solution indicate dynamic NMR because of restricted rotation around the carbon–nitrogen bond, resulting from conjugation of the side-chain nitrogen with the adjacent α,β-unsaturated ester group.

Molecular modeling of the voltammetric oxidation at a glassy carbon electrode of the antimalarial drug primaquine and its prodrugs succinylprimaquine and maleylprimaquine

The 8-aminoquinoline primaquine (PQ) is the only antimalarial drug used as tissue schizonticide and relapsing malaria. Antichagasic activity was also reported. Nevertheless, as it also shows serious side effects, prodrugs such as succinyl and maleyl derivatives have been proposed to decrease its toxicity. Although PQ mechanism of action has not been completely elucidated, the promotion of oxidative stress is an advanced hypothesis that could explain its activity in both plasmodia and trypanosome parasites. The oxidation of PQ and its prodrugs, maleylprimaquine (MPQ) and succinylprimaquine (SPQ), was studied by cyclic voltammetry using glassy carbon electrode. All compounds were oxidized in aqueous medium, with the charge transfer process being pH-dependent in acidic medium and pH-independent in a weak basic medium, being the neutral form more easily oxidized. This indicated that the protonation of the nitrogen atoms displays a determinant role in the voltammetric oxidation, being both prodrugs more easily oxidized than PQ protonated forms, in the order: SPQ < MPQ < PQ. For a better understanding of this behavior, a molecular modeling study was performed using the AM1 semi-empirical method from Spartan 04 for Linux (v.119, Wavefunction Inc.). The medium pH showed to be fundamental not only to the electronic density of the quinoline ring but also to the rearrangement of the nitrogen side chain. The electronic density of primaquine non-protonated quinoline ring is higher than that in its protonated and diprotonated species. Also, the use of prodrugs and the degree of saturation of the carriers (maleic or succinic acid) interfere with this feature. SPQ and MPQ have a slight increase in the quinoline electronic density in comparison to PQ. Nevertheless, the carrier in the side chain of SPQ is closer to the quinoline ring than it is in MPQ, which accounts for the higher electronic density in the former. The most significant effect occurs in the correspondent protonated forms of the nitrogen quinoline. The application of molecular modeling study associated to voltammetric techniques showed to be an important way to understand the redox mechanism of electroactive drugs. These results may be related to a biological activity and can be useful to future primaquine derivatives design.

Homonuclear and Heteronuclear NMR Studies of a Statherin Fragment Bound to Hydroxyapatite Crystals

Acidic proteins found in mineralized tissues act as nature's crystal engineers, where they play a key role in promoting or inhibiting the growth of minerals such as hydroxyapatite (HAP), Ca10(PO4)6(OH)2, the main mineral component of bone and teeth. Key to understanding the structural basis of protein-crystal recognition and protein control of hard tissue growth is the nature of interactions between the protein side chains and the crystal surface. In an earlier work we have measured the proximity of the lysine (K6) side chain in an SN-15 peptide fragment of the salivary protein statherin adsorbed to the Phosphorus-rich surface of HAP using solid-state NMR recoupling experiments. 15N{31P} rotational echo double resonance (REDOR) NMR data on the side-chain nitrogen in K6 gave rise to three different models of protein-surface interaction to explain the experimental data acquired. In this work we extend the analysis of the REDOR data by examining the contribution of interactions between surface phosphorus atoms to the observed 15N REDOR decay. We performed 31P-31P recoupling experiments in HAP and (NH4)2HPO4 (DHP) to explore the nature of dipolar coupled 31P spin networks. These studies indicate that extensive networks of dipolar coupled 31P spins can be represented as stronger effective dipolar couplings, the existence of which must be included in the analysis of REDOR data. We carried out 15N{31P} REDOR in the case of DHP to determine how the size of the dephasing spin network influences the interpretation of the REDOR data. Although use of an extended 31P coupled spin network simulates the REDOR data well, a simplified 31P dephasing system composed of two spins with a larger dipolar coupling also simulates the REDOR data and only perturbs the heteronuclear couplings very slightly. The 31P-31P dipolar couplings between phosphorus nuclei in HAP can be replaced by an effective dipolar interaction of 600 Hz between two 31P spins. We incorporated this coupling and applied the above approach to reanalyze the 15N{31P} REDOR of the lysine side chain approaching the HAP surface and have refined the binding models proposed earlier. We obtain 15N-31P distances between 3.3 and 5 A from these models that are indicative of the possibility of a lysine-phosphate hydrogen bond.

Biochemical Characterization and Crystal Structure of Synechocystis Arogenate Dehydrogenase Provide Insights into Catalytic Reaction

The extreme diversity in substrate specificity, and in the regulation mechanism of arogenate/prephenate dehydrogenase enzymes in nature, makes a comparative structural study of these enzymes of great interest. We report here on the biochemical and structural characterization of arogenate dehydrogenase from Synechocystis sp. (TyrAsy). This work paves the way for the understanding of the structural determinants leading to diversity in substrate specificity, and of the regulation mechanisms of arogenate/prephenate dehydrogenases. The overall structure of TyrAsy in complex with NADP was refined to 1.6 A. The asymmetric unit contains two TyrAsy homodimers, with each monomer consisting of a nucleotide binding N-terminal domain and a particularly unique alpha-helical C-terminal dimerization domain. The substrate arogenate was modeled into the active site. The model of the ternary complex enzyme-NADP-arogenate nicely reveals at the atomic level the concerted mechanism of the arogenate/prephenate dehydrogenase reaction.

A Chemical Switch for the Modulation of the Functional Activity of Higher Homologues of Histamine on the Human Histamine H3Receptor: Effect of Various Substitutions at the Primary Amino Function

In an effort to establish the structural requirements for agonism, neutral antagonism, and inverse agonism at the human histamine H(3) receptor (H(3)R) we have prepared a series of higher homologues of histamine in which the terminal nitrogen of the side chain has been either mono- or disubstituted with several aliphatic, alicyclic, and aromatic moieties or incorporated in cyclic systems. The novel ligands have been pharmacologically investigated in vitro for their affinities on the human H(3)R and H(4)R subtypes by radioligand displacement experiments and for their intrinsic H(3)R activities via a CRE-mediated beta-galactosidase reporter gene assay. Subtle changes of the substitution pattern at the side chain nitrogen alter enormously the pharmacological activity of the ligands, resulting in a series of compounds with a wide spectrum of pharmacological activities. Among the several neutral H(3)R antagonists identified within this series, compounds 2b and 2h display an H(3)R affinity in the low nanomolar concentration range (pK(i) values of 8.1 and 8.4, respectively). A very potent and selective H(3)R agonist (1l, pEC(50) = 8.9, alpha = 0.94) and a very potent, though not highly selective, H(3)R inverse agonist (2k, pIC(50) = 8.9, alpha = -0.97) have been identified as well.

Primary steps of the photochemical reactions of 2-cyano-10-(3-[dimethylamino, N-oxide]-2-methylpropyl)-5-oxide-phenothiazine, the photoproduct of cyamemazine, a phototoxic neuroleptic: comparison with the sulfoxide

The UVA-absorbing photoproduct resulting from the oxidation of the sulfur atom and of the side chain nitrogen of the phototoxic drug cyamemazine (CMZ) (2-cyano-10-(3-[dimethylamino]-2 methylpropyl)-phenothiazine) is a potent photodynamic photosensitizer. The photophysical and photochemical properties of this photoproduct (P) (2-cyano-10-(3-[dimethylamino, N-oxide]-2-methylpropyl)-5-oxide-phenothiazine)) have been investigated in neutral buffered aqueous solutions and in ethanol and compared to those of the sulfoxide (S) (2-cyano-10-(3-[dimethylamino]-2 methylpropyl)-5-oxide-phenothiazine), a CMZ oxidation product of cells. The fluorescence quantum yield (PhiF) of P is 0.25 and 0.21 in pH 7 phosphate buffer and ethanol, respectively. By contrast, S (PhiF = 0.14 in buffer) is practically unfluorescent in alcohol. In buffer, the fluorescence lifetimes of P and S are 10.5 and 11.8 ns, respectively. The transient absorbance of the first excited triplet state (3P1) with a characteristic absorption band peaking at 660 nm (epsilon = 5,300 M(-1) cm(-1)) has been observed by 355 nm laser flash spectroscopy of deaerated phosphate buffer or ethanol solutions. In buffer, the 3P1 lifetime is 0.5 micros. The energy transfer which occurs from the 3P1 to naproxen suggests that the 3P1 energy is greater than 62 kcal mol(-1). Triplet quenching by dioxygen occurs at rate 2.3 x 10(9) M(-1) s(-1). With the triplet benzophenone as actinometer, the 3P1 formation quantum yield is found to be 0. 40 in buffer. The 3P1 state is quenched by ethanol and 2-propanol with bimolecular reaction rate constants of 1.6 and 2.4 x 10(6) M(-1) s(-1), respectively. In buffer, P and S triplet states react with tryptophan, indole and cysteine at rate constants of the order of 10(9) M(-1) s(-1) for Trp and indole and 10(8) M(-1) s(-1) for Cys.

Mitochondrial toxicity of drugs

The introduction of this thesis concentrates on the cellular energy supplier and an important instrument in mediating cell death, mitochondrion. First, an overview is given to explain the biochemical properties of this organelle. Then, the role of mitochondria in cell death is discussed, followed by an article about the mitochondrial toxicity of drugs. As an example of the mitochondrial toxicity of a drug, an article about statins and their effects on L6 myocytes and rat muscle mitochondria conclude the introduction part of this thesis. The aim of the first project was to compare hepatocellular toxicity and pharmacological activity of amiodarone (B2-O-Et-N-diethyl) and eight amiodarone derivatives, including three amiodarone metabolites (B2-O-Et-NH-ethyl, B2-O-Et-NH2 and B2-O-Et-OH). In addition, five amiodarone analogues were investigated (B2-O-Et-N-dimethyl, B2-O-Et-N-dipropyl, B2-OAcetate, B2-O-Et-propionamide and B2-O-Et). The studies were accomplished using frehly isolated rat liver mitochondria, primary rat hepatocytes and the hepatoma cell line HepG2. The hepatocellular toxicity of amiodarone and most of the derivatives was confirmed. Amiodarone and most analogues showed a dose-dependent toxicity on the respiratory chain and on β- oxidation of the mitochondria. The ROS concentration in hepatocytes increased timedependently and apoptotic/necrotic cell populations were identified using flow cytometry and annexinV/propidiumiodide staining. The effect of the three least toxic amiodarone analogues on the hERG channel was compared to amiodarone. In conclusion, three amiodarone analogues (B2-O-Et-N-dipropyl, B2-O-Acetate and B2-O-Et) showed a lower hepatocellular toxicity profile than amiodarone and two of these analogues (B2-O-Et-N-dipropyl and B2-O-Acetate) retained hERG channel interaction capacity, suggesting that amiodarone analogues with class III antiarrhythmic activity and lower hepatic toxicity could be developed. For the second project in this thesis, we synthesized three more amiodarone analogues (B2-O-Ethylacetate, B2-O-Et-N-pyrrolidine and B2-O-Et-N-piperidine) and, together with amiodarone and its metabolites (B2-O-Et-NH-ethyl and B2-O-Et-NH2) and some derivatives from the first study (B2-O-Et-N-dipropyl, B2-O-Et-propionamide, B2-O-Acetate, B2-O-Et-OH), characterized their hepatic toxicity together with the pulmonary toxicity. The interaction with the hERG channel was determined for all the derivatives. Compared to amiodarone, which showed only a weak cytotoxicity, the desethylated metabolites, B2-O-Acetate, B2-O-Et-OH and B2-O-Et- N-pyrrolidine showed a similar or higher cytotoxicity. On the other hand, B2-O-Et-N-dipropyl, B2-O-Ethylacetate, B2-O-Et, B2-O-Et-propionamide and B2-O-Et-N-piperidine were less toxic. Cytotoxicity was associated with a drop in the mitochondrial membrane potential and therefore most probably mitochondrial in origin. Substances carrying a nitrogen in the side chain (amiodarone, B2-O-Et-NH-ethyl, B2-O-Et-NH2, B2-O-Et-N-dipropyl, B2-O-Et-propionamide, B2-O-Et-N-pyrrolidine, B2-O-Et-N-piperidine) showed a much higher affinity to the hERG channel (range 0.22-12.2µmol/L) than those without a nitrogen in this position (B2-O-Acetate, B2-OEthylacetate, B2-O-Et-OH) (range 74-216µmol/L). Neither cytotoxicity, nor the interaction with the hERG channel, was associated with the lipophilicity of the compounds. It was concluded, that the physicochemical properties of amiodarone and its analogues were not as important for the potassium channel interaction and cytotoxicity as the chemical structure of the compounds. In the third project of this thesis, the relationship between an unexpected toxicity of a drug and an underlying mitochondrial defect was studied using human dermal fibroblasts. These cells were derived from patients suffering from a mitochondrial defect. Simvastatin and benzbromarone are known to cause an unexpected adverse reaction (myotoxicity or hepatotoxicity, respectively). Both dermal fibroblasts with a mitochondrial defect and fibroblasts from healthy patients were treated with different concentrations of benzbromarone and simvastatin, and the overall toxicity was evaluated after different time points. There were no differences in the toxicity pattern between the cell lines, and the toxicity assayed was relatively scarce in all experiments. It was concluded that the the test system was not suitable for these studies and that they should be repeated with other cell lines of hepatic or muscle origin.

Antimuscarinic actions of antihistamines on the heart

Antimuscarinic side-effects, which include dry mouth, tachycardia, thickening of mucus possibly sedation, of the antihistamines limited the usefulness of these drugs. The advent of newer agents has reduced the sedative effect of the antihistamine. The data presented here show that one of the newest antihistamines, desloratadine, and a first generation drug, diphenhydramine, are both competitive inhibitors of muscarinic receptor mediated slowing of the heart as measured using a Langendorff preparation. Both agents have apparent sub-micromolar affinities for the muscarinic receptor. Two other agents, cetirizine and fexofenadine, do not interact with muscarinic receptors in the heart at the concentrations used in this study. Structural similarities of the drugs suggest that substitution of a group with a high dipole moment or charge on the side chain nitrogen decreases the binding with muscarinic receptors. We conclude that of the compounds tested fexofenadine and cetirizine have little or no interaction with muscarinic receptors.

Residues Distant from the Active Site Influence Protein-tyrosine Phosphatase 1B Inhibitor Binding

Regions of protein-tyrosine phosphatase (PTP) 1B that are distant from the active site yet affect inhibitor binding were identified by a novel library screen. This screen was based on the observation that expression of v-Src in yeast leads to lethality, which can be rescued by the coexpression of PTP1B. However, this rescue is lost when yeast are grown in the presence of PTP1B inhibitors. To identify regions of PTP1B (amino acids 1-400, catalytic domain plus 80-amino acid C-terminal tail) that can affect the binding of the difluoromethyl phosphonate (DFMP) inhibitor 7-bromo-6-difluoromethylphosphonate 3-naphthalenenitrile, a library coexpressing PTP1B mutants and v-Src was generated, and the ability of yeast to grow in the presence of the inhibitor was evaluated. PTP1B inhibitor-resistant mutations were found to concentrate on helix alpha7 and its surrounding region, but not in the active site. No resistant amino acid substitutions were found to occur in the C-terminal tail, suggesting that this region has little effect on active-site inhibitor binding. An in-depth characterization of a resistant substitution localizing to region alpha7 (S295F) revealed that this change minimally affected enzyme catalytic activity, but significantly reduced the potency of a panel of structurally diverse DFMP PTP1B inhibitors. This loss of inhibitor potency was found to be due to the difluoro moiety of these inhibitors because only the difluoro inhibitors were shifted. For example, the inhibitor potency of a monofluorinated or non-fluorinated analog of one of these DFMP inhibitors was only minimally affected. Using this type of library screen, which can scan the nearly full-length PTP1B sequence (catalytic domain and C-terminal tail) for effects on inhibitor binding, we have been able to identify novel regions of PTP1B that specifically affect the binding of DFMP inhibitors.

14 Modification of glutamine residues in proteins involved in translation

The only known protein glutamine methylations take place on proteins related to the ribosomal function. The modification is a mono-methylation on the side-chain amide nitrogen (N5). The glutamine residue within a Gly-Gly-Gln (GGQ) motif of the polypeptide release factor (RF) is methylated, and the modification is involved in polypeptide chain termination. A defect in RF methylation results in a dramatic growth defect and substantial read-through of stop codons during translation. Ribosomal protein L3 is also methylated on a glutamine residue. This modification is only found in bacteria and is not essential, but may be involved in efficient assembly of the ribosome structure. Gin methyltransferases (MTase) adopt the canonical class I MTase structure and contain a [D/N]PPY motif to position the substrate and facilitate catalysis. This motif was previously only thought to function in DNA N-MTases, but has been shown to act more generally on neutral planar amidesubstrates.