Effects of nicotinamide on NAD and poly (ADP-ribose) metabolism in DNA-damaged human lymphocytes

The effect of nicotinamide on unscheduled DNA synthesis was studied in resting human lymphocytes. In cells treated with UV irradiation or with MNNG, nicotinamide caused a two-fold stimulation of unscheduled DNA synthesis and retarded the rate of NAD⁺ lowering caused by these treatments. Nicotinamide also reduced the burst of poly(ADP-ribose) synthesis caused by MNNG treat-ment. Thus under conditions that it enhances unscheduled DNA synthesis, nicotinamide causes marked effects on the metabolism of NAD⁺ and poly(ADP-ribose). The effect of nicotinamide on unscheduled DNA synthesis was shown to be independent of protein or polyamine synthesis.     

Complexation of lead(II) by nicotinamide (nia): Crystal structure of polymeric Pb(nia)(NO3)2

Lead(II) nitrate complex with nicotinamide has been synthesized and characterized by elemental analysis, IR, ¹H NMR, ¹³C NMR, UV-Vis spectroscopy and X-ray crystallography. The Pb²⁺ centres linked through nicotinamide and nitrate(V) ligands form a two-dimensional polymeric structure defining a double layer and each layer is also stabilized by hydrogen bonds of N-H?O and C-H?O type.     

Flavin-nicotinamide biscoenzymes: models for the interaction between NADH (NADPH) and flavin in flavoenzymes. Reaction rates and physicochemical properties of intermediate species.

1. Flavin-nicotinamide biscoenzymes covalently linked by two, three or four methylene groups through positions N(10) of the flavin (Fl) and (N1) of the nicontinamide (Nic) form long-wavelength-absorbing, intramolecular complexes when the flavin part of the molecule is reduced specifically. The energy of the long-wavelength transition is minimal and its intensity maximal for (see journal for formula). 2. The increasing proximity of the positively charged nicotinamide lowers the pK-value of dihydroflavin deprotonation up to 1.7 units and the flavin oxidation-reduction potential becomes more positive up to 116 mV. 3. Specific reduction of the nicotinamide part of the biscoenzymes yields transient, long-wavelength-absorbing complexes. The energy of the long-wavelength transition is minimal and its intensity maximal for the complex (see journal for formula). 4. The rate of intramolecular flavin-dependent dihydronicotinamide dehydrogenation is highest for (see journal for formula), about 3 times slower for (see journal for formula) and 100 times slower for (see journal for formula). 5. The results obtained in this study are consistent with a reaction mechanism that involves formation of a charge transfer complex between reduced nicotinamide and oxidized flavin and rate-limiting heterolytic breakdown into products.     

Inhibition of nicotinamide phosphoribosyltransferase (NAMPT) activity by small molecule GMX1778 regulates reactive oxygen species (ROS)-mediated cytotoxicity in a p53- and nicotinic acid phosphoribosyltransferase1 (NAPRT1)-dependent manner

Cancer cells undergo mitosis more frequently than normal cells and thus have increased metabolic needs, which in turn lead to higher than normal reactive oxygen species (ROS) production. Higher ROS production increases cancer cell dependence on ROS scavenging systems to balance the increased ROS. Selectively modulating intracellular ROS in cancers by exploiting cancer dependence on ROS scavenging systems provides a useful therapeutic approach. Essential to developing these therapeutic strategies is to maintain physiologically low ROS levels in normal tissues while inducing ROS in cancer cells. GMX1778 is a specific inhibitor of nicotinamide phosphoribosyltransferase, a rate-limiting enzyme required for the regeneration of NAD(+) from nicotinamide. We show that GMX1778 increases intracellular ROS in cancer cells by elevating the superoxide level while decreasing the intracellular NAD(+) level. Notably, GMX1778 treatment does not induce ROS in normal cells. GMX1778-induced ROS can be diminished by adding nicotinic acid (NA) in a NA phosphoribosyltransferase 1 (NAPRT1)-dependent manner, but NAPRT1 is lost in a high frequency of glioblastomas, neuroblastomas, and sarcomas. In NAPRT1-deficient cancer cells, ROS induced by GMX1778 was not susceptible to treatment with NA. GMX1778-mediated ROS induction is p53-dependent, suggesting that the status of both p53 and NAPRT1 might affect tumor apoptosis, as determined by annexin-V staining. However, as determined by colony formation, GMX1778 long term cytotoxicity in cancer cells was only prevented by the addition of NA to NAPRT1-expressing cells. Exposure to GMX1778 may be a novel way of inducing ROS selectively in NAPRT1-negative tumors without inducing cytotoxic ROS in normal tissue.     

Interaction of lead(II) with beta-cyclodextrin in alkaline solutions

Polarographic and UV-spectrophotometric investigations of Pb(II) complex formation with beta-cyclodextrin have showed that the complexation of Pb(II) ions begins at pH >10. The formation of lead(II) 1:1 complex with the beta-cyclodextrin anion was observed at pH 10-11.5. The logarithm of the stability constant of this complex compound is 15.9+/-0.3 (20 degrees C, ionic strength 1.0), and the molar extinction coefficient value is ca. 5500 (lambda(max)=260 nm). With further increase in solution pH the Pb-beta-cyclodextrin complex decomposes and converts to Pb(OH)(2) or Pb(OH)(3)(-) hydroxy-complexes. This process occurs with a decrease in Pb(II) complexation degree. The latter result could be explained by a decrease in the beta-cyclodextrin anion activity. Neither Pb(OH)(2) nor Pb(OH)(3)(-) encapsulation into beta-CD cavity was observed.     

Formation of oligonuclear complexes between copper(II) and 1-hydroxyethane-1,1-diphosphonic acid

The complex formation between Cu(II) and 1-hydroxyethane-1,1-diphosphonic acid was studied by means of pH-potentiometry and spectroscopy (UV-Vis and EPR). Speciation revealed the formation of a trinuclear complex [Cu₃A₃]⁶⁻ besides the mononuclear 1:1 and 1:2 species. In the complex [Cu₃A₃]⁶⁻ , which is not formed with other diphosphonic derivatives, the ligands adopt chelating and bridging modes via the four O atoms of the two phosphonates, the alcoholic-OH groups remaining in the protonated form.     

Coenzyme binding and hydride transfer in Rhodobacter capsulatus ferredoxin/flavodoxin NADP(H) oxidoreductase

Ferredoxin-NADP(H) reductases catalyse the reversible hydride/electron exchange between NADP(H) and ferredoxin/flavodoxin, comprising a structurally defined family of flavoenzymes with two distinct subclasses. Those present in Gram-negative bacteria (FPRs) display turnover numbers of 1-5 s(-1) while the homologues of cyanobacteria and plants (FNRs) developed a 100-fold activity increase. We investigated nucleotide interactions and hydride transfer in Rhodobacter capsulatus FPR comparing them to those reported for FNRs. NADP(H) binding proceeds as in FNRs with stacking of the nicotinamide on the flavin, which resulted in formation of charge-transfer complexes prior to hydride exchange. The affinity of FPR for both NADP(H) and 2'-P-AMP was 100-fold lower than that of FNRs. The crystal structure of FPR in complex with 2'-P-AMP and NADP(+) allowed modelling of the adenosine ring system bound to the protein, whereas the nicotinamide portion was either not visible or protruding toward solvent in different obtained crystals. Stabilising contacts with the active site residues are different in the two reductase classes. We conclude that evolution to higher activities in FNRs was partially favoured by modification of NADP(H) binding in the initial complexes through changes in the active site residues involved in stabilisation of the adenosine portion of the nucleotide and in the mobile C-terminus of FPR.     

Inhibitory properties of N-nicotinyl-N' (substituted) urea analogs of NAD.

A series of N-nicotinyl- and N-isonicotinyl-N′-(substituted) ureas were synthesized by a condensation of the appropriate amide with various alkyl or aryl isocyanates, and examined as potential antimetabolites of nicotinamide. No significant microbial toxicity was observed; however, several derivatives in the nicotinyl series proved to be toxic to fish, especially N-nicotinyl-N′-hexylurea which possessed a TL₅₀ of 27 μg/ml. The toxicity was reversed by supplements of nicotinamide but not tryptophan, and may be due to the formation of an inactive NAD complex through the mediation of NADase. Evidence for this possibility is presented by the formation in vitro of an N-nicotinyl[7-¹⁴C]N′-ethylurea-NAD analog which was characterized through spectral, chemical, and chromatographic studies.     

Synthesis of Zn(II)-cloxacillin sodium complex and study of its interaction with calf thymus DNA

In this paper, a solid complex of cloxacillin sodium (CS) with Zn(II) was prepared by coprecipitation and characterized by UV, fluorescence, IR, and thermal spectra. Furthermore, the nature of the complex has been studied by ¹H-NMR and ¹³C-NMR spectroscopy. The influence of Zn(II) on the combination of CS and calf thymus DNA (CT DNA) was studied using fluorescence spectrophotometry, and the formation of binary CS-Zn(II) and CS-CT DNA complexes and ternary CS-Zn(II)-CT DNA complex was studied. The results show that the fluorescence intensity of CS can be quenched in the presence of Zn(II) or DNA. In the presence of Zn(II), the fluorescence quenching action of DNA on CS was strongly enhanced. Based on the fluorescence intensity, the formation constants of CS-Zn(II) and CS-CT DNA complexes were calculated, and the mechanism of interaction between CS, Zn(II), and DNA is discussed. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 2, pp. 184–193.     

Antitumor activity of phenylene bridged binuclear bis(imino-quinolyl)palladium(II) and platinum(II) complexes

Antitumor effects of a known bis(imino-quinolyl)palladium(II) complex 1 and its newly synthesized platinum(II) analogue 2 were evaluated against human breast (MCF-7) and human colon (HT-29) cancer cell lines. The complexes gave cytotoxicity profiles that were better than the reference drug cisplatin. The highest cytotoxic activities were pronounced in complex 2 across the two examined cancer cell lines. Both compounds represent potential active drugs based on bimetallic complexes.     

Crystal structure of NAD-dependent malate dehydrogenase complexed with NADP(H)

For better understanding of the coenzyme specificity in NAD-dependent MDH (tMDH) from Thermus flavus AT-62, we determined the crystal structures of tMDH-NADP(H) complex at maximally 1.65 A resolution. The overall structure is almost the same as that of the tMDH-NADH complex. However, NADP(H) binds to tMDH in the reverse orientation, where adenine occupies the position near the catalytic center and nicotinamide is positioned at the adenine binding site of the tMDH-NADH complex. Consistent with this, kinetic analysis of the malate-oxidizing reaction revealed that NADP(+) inhibited tMDH at high concentrations. This has provided the first evidence for the alternative binding mode of the nicotinamide coenzyme, that has pseudo-symmetry in its structure, in a single enzyme.     

Chitosan membranes modified by contact with poly(acrylic acid)

In this work chitosan membranes modified by contact with poly(acrylic acid) (PAA) aqueous solution at two different temperatures (25 °C and 60 °C) were obtained. The pure chitosan (CS) membranes, as well as those treated with PAA (CSPAA_25 and CSPAA_60) were characterized by FTIR-ATR, water sorption capacity, thermal analysis (TG/DTG), and scanning electron microscopy (SEM). In addition, in vitro permeation experiments were carried out using metronidazol and sodium sulfamerazine aqueous solutions at 0.1% and 0.2% as model drugs. FTIR-ATR results showed the presence of absorption bands of and COO⁻ indicating the formation of a polyelectrolyte complex between chitosan and poly(acrylic acid). The results also indicated that PAA penetrates deeper into the membrane at higher temperature (60 °C), forming a thicker complex layer. Polyelectrolyte complex formation as well as the influence of treatment temperature was confirmed by lower hydrophilicity, higher thermal stability, and lower permeability of the treated membranes. The results show that the methodology used is a simple and very efficient way to drastically change some membrane properties, especially their permeability.     

Human Lipopolysaccharide-binding Protein (LBP) and CD14 Independently Deliver Triacylated Lipoproteins to Toll-like Receptor 1 (TLR1) and TLR2 and Enhance Formation of the Ternary Signaling Complex

Bacterial lipoproteins are the most potent microbial agonists for the Toll-like receptor 2 (TLR2) subfamily, and this pattern recognition event induces cellular activation, leading to host immune responses. Triacylated bacterial lipoproteins coordinately bind TLR1 and TLR2, resulting in a stable ternary complex that drives intracellular signaling. The sensitivity of TLR-expressing cells to lipoproteins is greatly enhanced by two lipid-binding serum proteins known as lipopolysaccharide-binding protein (LBP) and soluble CD14 (sCD14); however, the physical mechanism that underlies this increased sensitivity is not known. To address this, we measured the ability of LBP and sCD14 to drive ternary complex formation between soluble extracellular domains of TLR1 and TLR2 and a synthetic triacylated lipopeptide agonist. Importantly, addition of substoichiometric amounts of either LBP or sCD14 significantly enhanced formation of a TLR1·TLR2 lipopeptide ternary complex as measured by size exclusion chromatography. However, neither LBP nor sCD14 was physically associated with the final ternary complex. Similar results were obtained using outer surface protein A (OspA), a naturally occurring triacylated lipoprotein agonist from Borrelia burgdorferi. Activation studies revealed that either LBP or sCD14 sensitized TLR-expressing cells to nanogram levels of either the synthetic lipopeptide or OspA lipoprotein agonist. Together, our results show that either LBP or sCD14 can drive ternary complex formation and TLR activation by acting as mobile carriers of triacylated lipopeptides or lipoproteins.     

A new ternary complex between horse liver alcohol dehydrogenase, NAD+, and acetone

Acetone was found to form a dead-end ternary complex with horse liver alcohol dehydrogenase and oxidized nicotinamide adenine dinucleotide (NAD⁺) when the reactants were incubated for a long time at relatively high concentrations. The complex formation was demonstrated by measuring the increase in absorbance at 320 nm, the quenching of protein fluorescence, and the loss of enzyme activity. Since acetone is a substrate of liver alcohol dehydrogenase, and the presence of acetaldehyde or pyrazole prevents acetone from forming the dead-end complex with liver alcohol dehydrogenase and NAD⁺, the acetone molecule in the complex may be bound to the substrate binding site of liver alcohol dehydrogenase. The dissociation of the complex was demonstrated by prolonged dialysis or by addition of reduced nicotinamide adenine dinucleotide (NADH) and iso-butyramide. A modified nicotinamide adenine dinucleotide was obtained as a main product from the dead-end complex after dissociation of the complex or denaturation of the apoenzyme. The modified nicotinamide adenine dinucleotide was found to exhibit an absorption spectrum similar to that of NADH; however, it was not oxidizable by liver alcohol dehydrogenase in the presence of acetaldehyde and exhibited no fluorescence.     

Equilibrium and kinetics of the dinuclear complex formation between N,N′-ethylenebis(salicylideneiminato)copper(II) and metal(II,I) ions in acetonitrile

The equilibrium constants and rate constants of the reactions between N,N′-ethylenebis(salicylideneiminato)copper(II) ([Cu(salen)]) and metal(II,I) ions in acetonitrile have been spectrophotometrically determined. [Cu(salen)] acts as a didentate ligand to form a dinuclear complex. The rate constants for the very labile Mn(II), Fe(II), and Zn(II) ions were directly evaluated using a variable flow-rate instrument that was newly constructed for this study. The rate constants of the dinuclear complex formation for a series of metal(II) ions vary in parallel with those of the acetonitrile solvent exchange on the corresponding metal(II) ions. This finding indicates that the dinuclear complex formation reaction of the metal(II) ions proceeds via almost the same reaction mechanism as for the acetonitrile solvent exchange reaction.     

Direct NAD(P)H hydrolysis into ADP-ribose(P) and nicotinamide induced by reactive oxygen species: a new mechanism of oxygen radical toxicity

The effect of different oxygen radical-generating systems on NAD(P)H was determined by incubating the reduced forms of the pyridine coenzymes with either Fe²⁺-H₂O₂ or Fe³⁺-ascorbate and by analyzing the reaction mixtures using a HPLC separation of adenine nucleotide derivatives. The effect of the azo-initiator 2,2'-azobis(2-methylpropionamidine)dihydrochloride was also tested. Results showed that, whilst all the three free radical-producing systems induced, with different extent, the oxidation of NAD(P)H to NAD(P)⁺, only Fe²⁺-H₂O₂ also caused the formation of equimolar amounts of ADP-ribose(P) and nicotinamide. Dose-dependent experiments, with increasing Fe²⁺ iron (concentration range 3-180 μM) or H₂O₂ (concentration range 50-1000 μM), were carried out at pH 6.5 in 50 mM ammonium acetate. NAD(P)⁺, ADP-ribose(P) and nicotinamide formation increased by increasing the amount of hydroxyl radicals produced in the medium. Under such incubation conditions NAD(P)⁺/ADP-ribose(P) ratio was about 4 at any Fe²⁺ or H₂O₂ concentration. By varying pH to 2.0, 3.0, 4.0, 4.5, 5.0, 5.5, 6.0, 7.0 and 7.4, NAD(P)⁺/ADP-ribose(P) ratio changed to 5.5, 3.2, 1.8, 1.6, 2.0, 2.5, 3.0, 5.4 and 6.5, respectively. Kinetic experiments indicated that 90-95% of all compounds were generated within 5s from the beginning of the Fenton reaction. Inhibition of ADP-ribose(P), nicotinamide and NAD(P)⁺ production of Fe²⁺-H₂O₂-treated NAD(P)H samples, was achieved by adding mannitol (10-50 mM) to the reaction mixture. Differently, selective and total inhibition of ADP-ribose(P) and nicotinamide formation was obtained by performing the Fenton reaction in an almost completely anhydrous medium, i.e. in HPLC-grade methanol. Experiments carried out in isolated postischemic rat hearts perfused with 50 mM mannitol, showed that, with respect to values of control hearts, this hydroxyl radical scavenger prevented reperfusion-associated pyridine coenzyme depletion and ADP-ribose formation. On the basis of these results, a possible mechanism of action of ADP-ribose(P) and nicotinamide generation through the interaction between NAD(P)H and hydroxyl radical (which does not involve the C-center where “conventional” oxidation occurs) is presented. The implication of this phenomenon in the pyridine coenzyme depletion observed in postischemic tissues is also discussed.     

Glutamine 132 in the NAD(H)-binding component of proton-translocating transhydrogenase tethers the nucleotides before hydride transfer

Transhydrogenase, found in bacterial membranes and inner mitochondrial membranes of animal cells, couples the redox reaction between NAD(H) and NADP(H) to proton translocation. In this work, the invariant Gln132 in the NAD(H)-binding component (dI) of the Rhodospirillum rubrum transhydrogenase was substituted with Asn (to give dI.Q132N). Mixtures of the mutant protein and the NADP(H)-binding component (dIII) of the enzyme readily produced an asymmetric complex, (dI.Q132N)(2)dIII(1). The X-ray structure of the complex revealed specific changes in the interaction between bound nicotinamide nucleotides and the protein at the hydride transfer site. The first-order rate constant of the redox reaction between nucleotides bound to (dI.Q132N)(2)dIII(1) was <1% of that for the wild-type complex, and the deuterium isotope effect was significantly decreased. The nucleotide binding properties of the dI component in the complex were asymmetrically affected by the Gln-to-Asn mutation. In intact, membrane-bound transhydrogenase, the substitution completely abolished all catalytic activity. The results suggest that Gln132 in the wild-type enzyme behaves as a "tether" or a "tie" in the mutual positioning of the (dihydro)nicotinamide rings of NAD(H) and NADP(H) for hydride transfer during the conformational changes that are coupled to the translocation of protons across the membrane. This ensures that hydride transfer is properly gated and does not take place in the absence of proton translocation.     

A kinetic analysis of cyanine selectivity: CCyan2 and Cyan40 intercalation into poly(dA-dT) x poly(dA-dT) and poly(dG-dC) x poly(dG-dC)

A T-jump investigation of the binding of Cyan40 [3-methyl-2-(1,2,6-trimethyl-4(1H)pyridinylidenmethyl)-benzothiazolium ion] and CCyan2 [3-methyl-2-[2-methyl-3-(3-methyl-2(3H)-benzothiazolylidene)-1-propenyl]-benzothiazolium ion] with poly(dA-dT) x poly(dA-dT) and poly(dG-dC) x poly(dG-dC) is performed at I = 0.1M (NaCl), 25 degrees C and pH 7. Two kinetic effects are observed for both systems. The binding process is discussed in terms of the sequence D + P <==> P,D <==> PD(I) <==> PD(II), which leads first to fast formation of a precursor complex P,D and then to a partially intercalated complex PD(I) which converts to the fully intercalate complex PD(II). Concerning CCyan2 the rate parameters depend on the polymer nature and their analysis shows that in the case of poly(dG-dC) x poly(dG-dC) the most stable bound form is the fully intercalated complex PD(II), whereas in the case of poly(dA-dT) x poly(dA-dT) the partially intercalated complex PD(I) is the most stable species. Concerning Cyan40, the rate parameters remain unchanged on going from A-T to G-C indicating that this dye is unselective.     

Structural and solution studies of a novel tetranuclear silver(I) cluster of [1,3-di(2-methoxy)benzene]triazene

The synthesis, characterization, crystal structure, ¹H NMR, spectrophotometric and conductometric studies of a new tetranuclear silver(I) complex of [1,3-di(2-methoxy)benzene]triazene are reported. Reaction of the ligand with silver acetate resulted in the formation of a tetranuclear cluster with a four-member Ag-Ag ring core, composed of four triazenide ligands and four metals. The results of studies of the stoichiometry and formation and of complex in tetrahydrofuran solution were found to be in support of its solid state structure.     

Chelating and bridging bis(diphenylphosphino)aniline complexes of copper(I)

The ligand bis(diphenylphosphino)aniline (dppan) has been shown to be a versatile ligand sporting different coordination modes and geometries as dictated by copper(I) and the counter ion. The molecular structures of its Cu(I) complexes were characterized by X-ray crystallography. The ligand was found in a chelating mode and monomeric complexes were formed when the ligand to copper ratio was 2:1 and the anion was non-coordinating. However, with thiocyanate as the counter anion, the ligand was found to adopt two different modes, with one ligand chelating and the other acting as a monodentate ligand. With CuX (X = Cl, Br), dppan formed a tetrameric complex when the ligand and metal were reacted in the ratio of 1:1. But reactions containing ligand and metal in the ratios of 1:2 or 2:1, resulted in the formation of a mixture of species in solution. Crystallization however, led to the isolation of the tetrameric complex. Variable temperature ³¹P{¹H} NMR spectra of the isolated tetramers did not show the presence of chelated structures in solution. Tetra-alkylammonium salts were added to solutions of various complexes of dppan and studied by ³¹P{¹H} NMR to probe the effect of anions on the stability of complexes in solution. The Cu-dppan complexes were robust and did not interconvert with other structures in solution unlike the bis(diphenylphosphino)isopropylamine complexes.