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PubMed2002

The ability of neonatal and maternal erythrocytes to produce reactive oxygen species in response to oxidative stress.

The ability of neonatal and maternal erythrocytes to produce reactive oxygen species (ROS) in response to oxidative stress was investigated using the chemiluminescence probe Cypridina luciferin analogue. The chemiluminescence probe, based on 2-methyl-6-[p-methoxyphenyl]-3,7-dihydro-imidazo[1,2-alpha]pyrazin-3-one (MCLA), is highly specific and sensitive to superoxide anion (O(2)(-)) and singlet oxygen (1O(2)). Blood from 11 mothers who experienced no complications and their healthy full-term newborns was collected and heparinized. MCLA was put into the washed erythrocytes suspension, and phenylhydrazine (PH) was added to cause oxidative stress. Chemiluminescence was measured using an Argus 50 image processing system. It was found that erythrocytes in neonatal blood had a 2.0-fold greater maximum chemiluminescence than did those in maternal blood. There was no change in the emission after the addition of NaN(3), but there was complete suppression with superoxide dismutase (SOD), demonstrating that these were O(2)(-). The present results demonstrated that neonatal erythrocytes produce about twice as much O(2)(-) as do adult erythrocytes in response to oxidative stress. With this method, it is very simple to measure the amount of O(2)(-) produced in erythrocytes and the rate at which they are produced. This method may therefore be very useful for determining the effects of antioxidants.

Adult↗

PubMed1998

pH regulation of the globular substance in the otoconial membrane of the guinea-pig inner ear.

Physiological and pharmacological characteristics of the globular substance, a precursor of otoconia, are not well understood. In the present study, we investigated the variations and regulation of internal pH (pHi) of the globular substance of the guinea-pig inner ear. The otoconial membrane was dissected out from the utricular macula and loaded with the pH-sensitive fluorophore, carboxyseminaphthorhodafluor-1. Dynamic changes of fluorescence were directly observed under a confocal laser scanning microscope, and pHi was calculated from dual emission ratio. In the NaCl standard solution buffered with 5 mM HEPES/Tris at pH 7.4, the pHi of the globular substance varied from 6.26 to 8.55 with an average of 7.21 (n = 270). Exposure to 25 mM NH+4 induced a rapid increase of the pHi followed by a slow relaxation. The, wash-out of NH+4 caused a prompt and pronounced acidification followed by a gradual pHi recovery to the initial level. This gradual pHi recovery was significantly inhibited by the absence of external Na+, indicating the presence of an external Na(+)-dependent H+ extrusion mechanism. This pHi recovery was also inhibited by 1 mM amiloride and 10 microM 3-amino-N-(aminoiminomethyl)-6-[ethyl(2-propyl)amino]pyrazine-2-ca rboxamide. These results suggest the presence of an Na(+)-H+ exchanger in the globular substance of the guinea pig. However, HCO3(-)-transporting mechanisms were not determined. The working hypothesis for the otoconial formation is discussed.

Acetazolamide↗

PubMed2000

A chemiluminescent assay for hydroperoxide level of phosphatidylcholine hydroperoxide fraction purified by two Sep-Pak cartridges in biological samples.

A chemiluminescent assay for hydroperoxide level of phosphatidylcholine hydroperoxide (PCOOH) fraction purified from biological samples was presented. This method utilized of two Sep-Pak cartridges. A lipid soluble fraction was isolated from each homogenized tissue or blood by Folch's method. The mixture of phosphatidylcholine (PC) and PCOOH was separated from the lipid soluble fraction by a Sep-Pak silica cartridge. A Sep-Pak tC18 cartridge made complete separation of both PCOOH and PC possible. The hydroperoxide level of PCOOH fraction was quantified by the reaction with ferrous ion using 2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazo[1,2-a]pyrazin++ +-3-one as a chemiluminescent dye. The mixture of positional isomers, 1-hexadecanoyl-2-[9, or 10-hydroperoxyl octadecanoyl]-sn-glycero-3-phosphocholine was used as an authentic standard. The good recovery rate for authentic PCOOH of 87.1 +/- 11.6% (mean +/- S.E., n = 4) was obtained by using two Sep-Pak cartridges. Linear calibration curve was obtained in the range from 2.5 to 20 nmol, and the detection limit of the standard was 10 pmol (signal-to-noise ratio > 3). This method was applied to the investigation of the lipid peroxidation induced by reperfusion of the liver with cold preservation, mimicking liver transplantation in rats. The effect of liposome-encapsulated dichloromethylene diphosphonate (LEDD), which eliminate of Kupffer cells to prevent the generation of oxygen radicals on the lipid peroxidation, was compared with the untreated group as a control. After 1 h reperfusion at 37 degrees C the hydroperoxide level obtained the liver without preservation in the untreated group was 12.4 +/- 2.4 nmol/100 mg lipid (n = 4) and levels increased significantly by prolongation of the preservation time. On the other hand, the hydroperoxide level in the LEDD treated group did not change up to 24 h preservation. These results suggest that this improved assay for hydroperoxide level of PCOOH fraction in biological samples can be applied to investigations involving lipid peroxidation because of its simplicity and accuracy.

Animals↗

PubMed2001

Reaction of tetrahydrobiopterin with superoxide: EPR-kinetic analysis and characterization of the pteridine radical.

It has been shown that BH(4) ameliorates endothelial dysfunction associated with conditions such as hypertension, cigarette smoking, and diabetes. This effect has been proposed to be due to a superoxide scavenging activity of BH(4). To examine this possibility we determined the rate constant for the reaction between BH(4) and superoxide using electron paramagnetic resonance (EPR) spin trapping competition experiments with 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO). We calculated a rate constant for the reaction between BH(4) and superoxide of 3.9 +/- 0.2 x 10(5) M(-1)s(-1) at pH 7.4 and room temperature. This result suggests that superoxide scavenging by BH(4) is not a major reaction in vivo. HPLC product analysis showed that 7,8-BH(2) and pterin are the stable products generated from the reaction. The formation of BH(4) cation radical (BH(4)(*+)) was demonstrated by direct EPR only under acidic conditions. Isotopic substitution experiments demonstrated that the BH(4)(*+) is mainly delocalized on the pyrazine ring of BH(4). In parallel experiments, we investigated the effect of ascorbate on 7,8-BH(2) reduction and eNOS activity. We demonstrated that ascorbate does not reduce 7,8-BH(2) to BH(4), nor does it stimulate nitric oxide release from eNOS incubated with 7,8-BH(2). In conclusion, it is likely that BH(4)-dependent inhibition of superoxide formation from eNOS is the mechanism that better explains the antioxidant effects of BH(4) in the vasculature.

Ascorbic Acid↗

PubMed1999

On topography and functionality in the B-D rings of cephalostatin cytotoxins.

Analogues 12'beta-hydroxycephalostatin 1 (9), 7'-deoxyritterazine G (10), and 14-epi-7'-deoxyritterazine B (11) were prepared via our protocol for unsymmetrical pyrazine synthesis. Cytotoxicity against human tumors was also determined for the first time for ritterazines, with femtomolar potency and a high correlation to cephalostatins observed. The SAR of these and related compounds provide insight into the importance of topography and certain chemical functionality in the B-D and B'-D' rings of cephalostatin type antineoplastics.

Antineoplastic Agents↗

PubMed1999

Exploratory solid-phase synthesis of factor Xa inhibitors: discovery and application of p3-heterocyclic amides as novel types of non-basic arginine surrogates.

A series of novel FXa inhibitors 2a-m and 3a-f was discovered that feature heterocyclic carboxamides tethered to a d-diaminobutyric acid sidechain. These neutral amide derivatives serve as novel P3 d-arginine mimics. Pyrazine carboxamide scaffolds afforded the most potent FXa inhibitors (e.g., 2b IC50 = 4.6 nM). The synthesis and biological activity of two focused libraries are reported.

Amides↗

PubMed2001

Synthesis and evaluation of diazine containing bioisosteres of (-)-ferruginine as ligands for nicotinic acetylcholine receptors.

In this structure-affinity relationship (SAFIR) study, the bioisosteric potential of diazines in the field of ferruginine-type nAChR ligands was investigated. Novel enantiopure analogues of (-)-Ferruginine (3) such as 6-8 were synthesized utilizing enantiomerically pure N-protected (+)-2-tropanone 9 from the 'chiral pool' as versatile chiral building block and a palladium-catalyzed Stille cross-coupling of the tributylstannyl diazines 12, 14 and 16 with the vinyl triflate 11 of (+)-2-tropanone 9. The structures of the novel diazine analogues 6-8 of (-)-ferruginine (3) were assigned on the basis of spectral data, that of ligand 7 being additionally verified by X-ray crystallography. The bioisosteric replacement of the acetyl moiety as structural part of the lead compound 3 with the pyridazine, pyrimidine and pyrazine nucleus resulted in ligands with high to moderate affinity for the central alpha4beta2 and remarkably low affinity for the alpha7* nAChR subtypes. Among the compounds synthesized and tested, 7 was the most active one with K(i)=3.7 nM (alpha4beta2). Compared with the lead 3, this value represents a 30-fold improvement in the affinity for the alpha4beta2 subtype combined with a substantially improved selectivity ratio between the alpha4beta2 and alpha7* subtypes.

Animals↗

PubMed2003

Semisynthesis of heterocyclic analogues of squamocin, a cytotoxic annonaceous acetogenin, by an unusual oxidative decarboxylation reaction.

In addition to two expected pyrazin derivatives, two imidazole analogues of squamocin 1 have been semisynthetised from squamocin derived alpha-ketoesters/alpha-ketoacid, via an unusual condensation-oxidative decarboxylation reaction with 1,2 diamines in presence of acetic acid and oxygen as the key step. Some of these analogues exhibited potent, although significantly reduced cytotoxicities relatively to squamocin 1. In addition, benzimidazole 8 possessed in comparison with the natural acetogenin some interesting cell cycle effects.

Cell Cycle↗

PubMed2004

Experimental studies on the determination of the dipole moments of some different laser dyes.

In this paper we used the Stokes shift phenomena to determine the difference in the dipole moments of the excited state micro(e) and ground state micro(g) to be (micro(e)-micro(g)), and the polarizability alpha. In this paper, we studied six different laser dyes belonging to four different classes of laser dyes which are diolefin 2,5-Distyryl pyrazine (DSB); 1,4-Bis (-pyridyl-2-vinyl) benzene (P2VB) and p-Bis-(o-methylstyryl)-benzene (Bis-MSB) with (micro(e)-micro(g)) equal to 6.40, 6.70 and 2.98 Debye, respectively; anthracene class includes 10(4-acetoxyphenyl)-2-methyl-9-acetoxy anthracene (APMAA) with (micro(e)-micro(g)) value of 7.25 Debye; Rhodamine B (RB) with (micro(e)-micro(g)) value of 5.33 Debye; and Coumarin 120 (C120) with the value 3.97 Debye for (micro(e)-micro(g)). In addition the value of both polarizability alpha and the radius r of each investigated laser dye molecule are determined. Therefore, the ratio alpha/r(3) for each dye is calculated to be 0.93, 0.79, 0.39, 0.37, 0.67 and 0.76 for DSP, P2VB, Bis-MSB, APMAA, RB and C120, respectively. The values of r are 4.83, 4.83, 4.90, 5.34, 5.75 and 4.11 A for the above consequence laser dyes. These dyes are studied in a large number of different solvents. The values obtained of (micro(e)-micro(g)) for these selected dyes are positive, which means that the excited state is more polar than the ground state.

Anthracenes↗

PubMed1998

Micromethod for the investigation of the interactions between DNA and redox-active molecules.

A novel microscale and surface-based method for the study of the interactions of DNA with other redox-active molecules using DNA-modified electrodes is described. The method is simple, convenient, reliable, reagent-saving, and applicable for DNA studies, especially those involving microsamples. Information such as binding site size (s, in base pairs), binding constant (K), ratio (K0x/KRed) of the binding constants for the oxidized and reduced forms of a bound species, binding free energy (delta Gb), and interaction mode, including changes in the mode of interaction, and "limiting" ratio K0x0/KRed0 at zero ionic strength can be obtained using only 3-15 micrograms of DNA samples. The method was developed using [Co(Phen)3]3+/2+ (Phen = 1,10-phenanthroline)/double-stranded DNA (dsDNA)-modified gold electrodes and [Co(bpy)3]3+/2+ (2,2'-bipyridyl)/dsDNA-modified gold electrodes as model systems. For the [Co(Phen)3]3+/2+/dsDNA-modified gold electrode system, a K2+ of (2.5 +/- 0.3) x 10(5) M-1 and an s of 5 bp were obtained in 5 mM pH 7.1 Tris-HCl buffer solution containing 50 mM NaCl. For [Co(bpy)3]3+/2+/dsDNA-modified gold electrodes, K3+ and s values of (1.3 +/- 0.3) x 10(5) M-1 and 3 bp, respectively, were obtained. While the s values are consistent with those reported in the literature obtained by solution methods, the K values are almost an order of magnitude larger. A transition in the nature of the interaction between dsDNA and [Co(Phen)3]3+/2+, from electrostatic to intercalative with increasing ionic strength, was found in our studies. Negative values of delta E0' for [Co(bpy)3]3+/2+ bound to dsDNA suggest that its interaction with dsDNA is predominantly electrostatic over the ionic strength range of 5-105 mM. The "limiting" ratio K3+0/K2+0 of 22 obtained for [Co(Phen)3]3+/2+ bound to dsDNA at zero ionic strength suggests that electrostatic interactions are predominant over intercalative ones under these limiting conditions. The ratio for [Co(bpy)3]3+/2+ of 16 also indicates that the 3+ form binds to dsDNA more strongly than the 2+ form at zero ionic strength. For [Co(Phen)3]3+/2+/single-stranded DNA (ssDNA)-modified gold electrodes, the nonuniformity of the surface structure of ssDNA-modified gold electrodes greatly complicates the analysis. A system consisting of a dsDNA-modified gold electrode and [Co(tppz)2]3+/2+ (tppz = tetra-2-pyridyl-1,4-pyrazine) was studied by this method, with a K2+ value of (5 +/- 1) x 10(5) M-1 and an 8 value of 7 bp being obtained.

2,2'-Dipyridyl↗

PubMed2002

Unsaturated O- and N-heterocycles from carbohydrate feedstocks.

With reference to the impending transition of chemical industry from depleting fossil raw materials to renewable feedstocks--the end of cheap oil is predicted for 2040 at the latest--this account gives an overview on chemically transforming carbohydrates, by far the major part of the annually regrowing biomass, into the following unsaturated O- and N-heterocycles with versatile industrial application profiles: furans, pyrans, dihydropyranones, pyrroles, pyrazoles, imidazoles, pyridinols, pyrazines, and quinoxalines. Although the emphasis was laid on conversions that can be effected in practical one-pot procedures or in a few large-scale-adaptable steps, a broad structural variety of products emerges that have not only diverse chemical functionalities but also hydrophilic "residual sugar" portions that render them water-soluble and readily biodegradable.

Biodegradation, Environmental↗

PubMed2005

New approaches to the analysis of high connectivity materials: design frameworks based upon 4(4)- and 6(3)-subnet tectons.

Coordination framework polymers derived from lanthanide metal ions with N,N'-dioxide ligands (4,4'-bipyridine-N,N'-dioxide, pyrazine-N,N'-dioxide, 1,2-bis(pyridin-4-yl)ethane-N,N'-dioxide, trans-1,2-bis(pyridin-4-yl)ethene-N,N'-dioxide) exhibit such intricate architectures that a new strategy is required to appreciate and understand their structures. Rather than analyzing the overall structure in terms of the connectivity of individual metal nodes, which can lead in some cases to extremely complex topological treatments, our new strategy is based on the visualization of the structures as combinations of interconnected layered 2-D sheets or subnet tectons. Despite the diversity and relative complexities of many of the structures discussed here, they can all be described by the interconnection of just two types of 2-D subnet tectons, 4(4) square grids or 6(3) hexagonal grids. The interconnection of these layered sheets with bridging N,N'-dioxide molecules gives rise to both 2-D bilayer and 3-D network extended structures depending upon the relative dispositions of the interconnecting N,N'-dioxide ligands. Thus, 2-D bilayers result when the N,N'-dioxide ligands that bridge two subnet tectons are located on the same side of the sheet, while 3-D networks are formed when the bridging N,N'-dioxide ligands are located on both sides of the sheet. This analysis allows ready identification and interpretation of some of the most highly connected and complex architectures yet observed in materials chemistry.

Journal Article↗

PubMed1985

Theoretical studies on the activation of the pterin cofactor in the catalytic mechanism of dihydrofolate reductase.

Two mechanisms for facilitating hydride ion transfer from NADPH involving preprotonation of the pteridine rings of the dihydrofolate reductase substrates folate and dihydrofolate have been investigated by ab initio quantum mechanical methods. Protonation energies and effective solution pKas have been calculated for four protonated forms, three of which are nonpreferred in aqueous solution and therefore not directly accessible to experimental study. The pattern and degree of redistribution of the positive charge over the component rings of the N-heterobicyclic pi-system in these protonated forms have been analyzed in terms of changes in the electron populations of the ring atoms and total ring charges. The effects of such changes in promoting hydride ion transfer to C7 in folate and C6 in dihydrofolate have been evaluated by considering the extent of development of partial carbonium ion character at these carbon atoms and also the degree of electron deficiency in the pyrazine ring as a whole. The results illustrate that perturbations due, for instance, to protonation may be propagated by pi-electron coupling effects over medium-range distances of 4-6 A across the pteridine ring. The two mechanisms have been assessed in terms of the calculated absolute and relative pKas of the protonated species taking into account experimental information regarding possible stabilization of these forms in the enzyme active site and also the effectiveness of the various protonations in assisting the hydride ion transfer step. Judged against these criteria, the theoretical results favor the generally proposed mechanism involving preprotonation of N8 in folate and N5 in dihydrofolate. However, some support was also found for the alternative novel mechanism involving O4-protonation of both folate and dihydrofolate.

Calorimetry↗

PubMed1986

Pyrimidodiazepine, a ring-strained cofactor for phenylalanine hydroxylase.

Homologues of 6-methyl-7,8-dihydropterin (6-Me-7,8-PH2) and 6-methyl-5,6,7,8-tetrahydropterin (6-Me-PH4), expanded in the pyrazine ring, were synthesized to determine the effect of increased strain on the chemical and enzymatic properties of the pyrimidodiazepine series. 2-Amino-4-keto-6-methyl-7,8-dihydro-3H,9H-pyrimido[4,5-b] [1,4]diazepine (6-Me-7,8-PDH2) was found to be more unstable in neutral solution than 6-Me-7,8-PH2. Its decomposition appears to proceed by hydrolytic ring opening of the 5,6-imine bond, followed by autooxidation. 6-Me-7,8-PDH2 can be reduced, either chemically or by dihydrofolate reductase (Km = 0.16 mM), to the 5,6,7,8-tetrahydro form (6-Me-PDH4). This can be oxidized with halogen to quinoid dihydropyrimidodiazepine (quinoid 6-Me-PDH2), which is a substrate for dihydropteridine reductase (Km = 33 microM). Whereas quinoid 6-methyldihydropterin was found to tautomerize to 6-Me-7,8-PH2 in 95% yield in 0.1 M tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), pH 7.4, quinoid 6-Me-PDH2 gives only 53% 6-Me-7,8-PDH2, the remainder decomposing via an initial opening of the diazepine ring. Additional evidence for the extra strain in the pyrimidodiazepine system is the cyclization of quinoid 6-N-(2'-aminopropyl)divicine to quinoid 6-Me-PH2 in 57% yield in 0.1 M Tris-HCl, pH 7.4. By comparison, no quinoid 6-Me-PDH2 is formed from the homologue quinoid 6-N-(3'-aminobutyl)divicine. A small (2%) yield of 6-Me-PDH4 is found if the unstable C4a-carbinolamine intermediate is trapped by enzymatic dehydration and reduction. Although phenylalanine hydroxylase utilizes 6-Me-PDH4 (Km = 0.15 mM), the maximum velocity of tyrosine production is 20 times slower than that with 6-Me-PH4, indicating that a ring opening reaction is not a rate-limiting step in the hydroxylase pathway. Further, the maximum velocities of 2,5,6-triamino-4(3H)-pyrimidinone, 2,6-diamino-5-(methylamino)-4(3H)-pyrimidinone, and 2,6-diamino-5-(benzylamino)-4(3H)-pyrimidinone span a 35-fold range. These cofactors would theoretically form the same oxide of quinoid divicine if oxygen activation involves a carbonyl oxide intermediate. Thus, the limiting step is also not transfer of oxygen from this hypothetical intermediate to the phenylalanine substrate.

Animals↗

PubMed1987

Adduct formation between the cupric site of phenylalanine hydroxylase from Chromobacterium violaceum and 6,7-dimethyltetrahydropterin.

The interaction of pterin-dependent phenylalanine hydroxylase from Chromobacterium violaceum with the cofactor analogue 5-deaza-6-methyltetrahydropterin and the cofactor 6,7-dimethyltetrahydropterin (DMPH4) has been investigated by multifrequency electron spin resonance (ESR) spectroscopy. 5-Deaza-6-methyltetrahydropterin, which lacks the N-5 nitrogen present in the pyrazine ring of DMPH4, binds tightly to the cupric form of the enzyme; however, no changes are observed in the ESR parameters of the copper center. In contrast, the binding of DMPH4 (or 6-methyltetrahydropterin) shifts the ESR parameters (g and A) associated with the cupric enzyme. In addition, superhyperfine transitions were resolved and assigned to hyperfine splitting from nitrogen ligands. ESR spectra of the enzyme recorded in the presence of [5-14N]DMPH4 or [5-15N]DMPH4 were computer simulated and found to be consistent with pterin serving as a direct donor ligand to the copper center through the N-5 position.

Binding Sites↗

PubMed1981

Molecular complex of lumiflavin and 2-aminobenzoic acid: crystal structure, crystal spectra, and solution properties.

The molecular complex lumiflavin-2-aminobenzoic acid monohydrate (C13H12N4O2.C7H7NO2.H2O) crystallizes from from aqueous solution as red triclinic prisms. The space group is P1 with cell dimensions a = 9.660 A, b = 14.866 A, c = 7.045 A, alpha = 95.44 degrees , beta = 95.86 degrees, and gamma = 105.66 degrees . The crystal structure was solved by direct methods and refined by block-diagonal least-squares procedures to an R value of 0.050 on the basis of 1338 observed reflections. The structure is composed of stacks of alternating lumiflavin adn un-ionized (neutral) 2-aminobenzoic acid molecules. Two different modes of stacking interaction are observed. In one, 2-aminobenzoic acid overlaps all three of the isoalloxazine rings, at a mean distance of 3.36 A; in the other, 2-aminobenzoic acid interacts distance of 3.36 A; in the other, 2-aminobenzoic acid interacts with the pyrazine and dimethylbenzene moieties, at a distance of 3.42 A. Perpendicular to the stacking direction, the molecules form a continuous sheet. Each flavin is hydrogen bonded via O(2) and NH(3) to two symmetrically related aminobenzoates; the water of crystallization forms three hydrogen bonds, bridging two flavins, via O(4) and N(5), and one aminobenzoic acid. The red color of the crystals results from a charge-transfer transition involving stacked flavin and 2-aminobenzoic acid. The red color of the crystals results from a charge-transfer transition involving stacked flavin and 2-aminobenzoic acid molecules. Measurements of the polarized optical absorption spectra of crystals show that the transition moment direction for the long wavelength absorbance (beyond 530 nm) contains an out-of-plane component which can only arise from a charge-transfer interaction. Since the amino N does not make exceptionally close interactions with isoalloxazine atoms in either stacking mode (minimum interatomic distance 3.52 A), the charge transfer is presumed to involve pi orbitals of the 2-aminobenzoic acid donor.

Chemical Phenomena↗

PubMed2003

Chromophore-modified bisnaphthalimides: DNA recognition, topoisomerase inhibition, and cytotoxic properties of two mono- and bisfuronaphthalimides.

Bisnaphthalimides represent a promising group of DNA-targeted anticancer agents. In this series, the lead compounds elinafide and bisnafide have reached clinical trials, and the search for more potent analogues remains a priority. In the course of a medicinal chemistry program aimed at discovering novel antitumor drugs based on the naphthalimide skeleton, different dimeric molecules containing two tetracyclic neutral DNA intercalating chromophores were synthesized. The naphthalimide unit has been fused to a benzene ring (azonafide derivatives), an imidazole, a pyrazine, or, as reported here, a furan ring which increases the planar surface of the chromophore and enhances its stacking properties. We report a detailed investigation of the DNA binding capacity of the dimeric molecule MCI3335 composed of two furonaphthalimide units connected by a 12 A long amino alkyl linker [(CH(2))(2)-NH-(CH(2))(3)-NH-(CH(2))(2)] identical to that of elinafide. Qualitative and quantitative binding studies, in particular using surface plasmon resonance, establish that the dimer binds considerably more tightly to DNA (up to 1000 times) than the corresponding monomer and exhibits a higher sequence selectivity for GC-rich sequences. DNase I footprinting experiments attest that the dimer, and to a lesser extent the monomer, preferentially intercalate at GC sites. The strong binding interaction between the drugs and DNA perturbs the relaxation of supercoiled DNA by topoisomerases, but the test compounds do not promote DNA cleavage by topoisomerase I or II. Despite the lack of poisoning effect toward topoisomerase II, MCI3335 displays a very high cytotoxicity toward CEM human leukemia cells, with an IC(50) in the low nanomolar range, approximately 4 times inferior to that of the reference drug elinafide. Confocal microscopy observations indicate that the monomer shows a stronger tendency to accumulate in the cell nuclei than the dimer. The extremely high cytotoxic potential of MCI3335 is attributed to its enhanced capacity to bind to DNA and to inhibit DNA synthesis, as evidenced by flow cytometry experiments using the BrdU assay. The results provide novel mechanistic information that furthers the understanding of the structure-activity relationships in the bisnaphthalimide series and identify MCI3335 as a novel lead compound for further preclinical investigations.

Base Sequence↗

PubMed2004

Traditional GFP-type cyclization and unexpected fragmentation site in a purple chromoprotein from Anemonia sulcata, asFP595.

The purple chromoprotein (asFP595) from Anemonia sulcata belongs to the family of green fluorescent protein (GFP). Absorption and emission spectra of asFP595 are similar to those of a number of recently cloned GFP-like red proteins of the DsRed subfamily. The earlier proposed asFP595 chromophore structure [Martynov, V. I.; et al. (2001) J. Biol. Chem. 276, 21012-21016] was postulated to result from an "alternative cyclization" giving rise to a pyrazine-type six-membered heterocycle. Here we report that the asFP595 chromophore is actually very close in chemical structure to that of zFP538, a yellow fluorescent protein [Zagranichny, V. E.; et al. (2004) Biochemistry 43, 4764-4772]. NMR spectroscopic studies of four chromophore-containing peptides (chromopeptides) isolated under mild conditions from enzymatic digests of asFP595 and one chromopeptide obtained from DsRed revealed that all of them contain a p-hydroxybenzylideneimidazolinone moiety formed by Met-65/Gln-66, Tyr-66/67, and Gly-67/68 of asFP595/DsRed, respectively. Two asFP595 chromopeptides are proteolysis products of an isolated full-length polypeptide containing a GFP-type chromophore already formed and arrested at an earlier stage of maturation. The two other asFP595 chromopeptides were isolated as proteolysis products of the purified chromophore-containing C-terminal fragment. One of these has an oxo group at Met-65 C(alpha) and is a hydrolysis product of another one, with the imino group at Met-65 C(alpha). The N-unsubstituted imino moiety of the latter is generated by spontaneous polypeptide chain cleavage at a very unexpected site, the former peptide bond between Cys-64 C' and Met-65 N(alpha). Our data strongly suggest that both zFP538 and asFP595 could be attributed to the DsRed subfamily of GFP-like proteins.

Animals↗