DNA modification by 4-aza-3-ene-1,6-diynes: DNA cleavage, pH-dependent cytosine-specific interactions, and cancer cell cytotoxicity

The (Z)-hex-1,5-diyne-3-ene reactive core common to the enediyne antitumor antibiotics undergoes a Bergman cyclization after proper activation to afford reactive diradical intermediates that are responsible for initiating DNA cleavage. Direct modification of the enediyne core has been proposed as a method to permit cancer cell-specific triggering of the diradical-generating cyclization. For example, 3-aza-3-ene-1,5-diynes undergo an aza-Bergman cyclization to afford the fleeting 2,5-didehydropyridine diradicals. While protonation of these aza-enediynes can afford products of diradical trapping, the hydrolytic instability of the 3-aza-3-ene-1,5-diyne moiety prevents its use in pH-triggered DNA cleaving anticancer agents. Recently, more hydrolytically stable systems incorporating the 4-aza-3-ene-1,6-diyne moiety were developed. We report here studies of the 4-aza-3-ene-1,6-diyne-containing benzimidazolium salt AZB002 [1-methyl-2-(phenylethynyl)-3-(3-phenylprop-2-ynyl)-3H-benzimidazolium tetrafluoroborate] and two structurally related heterocycles that lack the aza-enediyne functionality, AZB016 [1,3-dimethyl-2-(phenylethynyl)-3H-benzimidazolium triflate] and AZB004 [3-methyl-2-(phenylethynyl)benzothiazolium triflate]. The interaction of these compounds with supercoiled DNA, a double-stranded DNA fragment, and a short DNA duplex oligonucleotide was investigated. There are three distinct DNA interactions exhibited by AZB002: a frank strand scission leading to the relaxation of supercoiled DNA and formation of at least two different DNA adducts, one of which leads to cytosine-specific cleavage after piperidine/heat treatment. In contrast, analogues lacking the aza-enediyne functionality either fail to interact with DNA (AZB016) or cleave DNA at guanine residues, presumably through alkylation of the N-7 position (AZB004). We also investigated the cytotoxicity of AZB002 and the related heterocyclic compounds AZB004 and AZB016 and find that only the DNA interactive compounds AZB002 and AZB004 display significant cytotoxicity. In particular, AZB002 is cytotoxic against a wide range of cancer cell lines.     

Reversal of the deoxyhypusine synthesis reaction. Generation of spermidine or homospermidine from deoxyhypusine by deoxyhypusine synthase

Deoxyhypusine synthase catalyzes the first step in hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine) synthesis in a single cellular protein, eIF5A precursor. The synthesis of deoxyhypusine catalyzed by this enzyme involves transfer of the 4-aminobutyl moiety of spermidine to a specific lysine residue in the eIF5A precursor protein to form a deoxyhypusine-containing eIF5A intermediate, eIF5A(Dhp). We recently discovered the efficient reversal of deoxyhypusine synthesis. When eIF5A([3H]Dhp), radiolabeled in the 4-aminobutyl portion of its deoxyhypusine residue, was incubated with human deoxyhypusine synthase, NAD, and 1,3-diaminopropane, [3H]spermidine was formed by a rapid transfer of the radiolabeled 4-aminobutyl side chain of the [3H]deoxyhypusine residue to 1,3-diaminopropane. No reversal was observed with [3H]hypusine protein, suggesting that hydroxylation at the 4-aminobutyl side chain of the deoxyhypusine residue prevents deoxyhypusine synthase-mediated reversal of the modification. Purified human deoxyhypusine synthase also exhibited homospermidine synthesis activity when incubated with spermidine, NAD, and putrescine. Thus it was found that [14C]putrescine can replace eIF5A precursor protein as an acceptor of the 4-aminobutyl moiety of spermidine to form radiolabeled homospermidine. The Km value for putrescine (1.12 mM) as a 4-aminobutyl acceptor, however, is much higher than that for eIF5A precursor (1.5 microM). Using [14C]putrescine as an acceptor, various spermidine analogs were evaluated as donor substrates for human deoxyhypusine synthase. Comparison of spermidine analogs as inhibitors of deoxyhypusine synthesis, as donor substrates for synthesis of deoxyhypusine (or its analog), and for synthesis of homospermidine (or its analog) provides new insights into the intricate specificity of this enzyme and versatility of the deoxyhypusine synthase reaction.     

The biosynthesis of hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine). Alignment of the butylamine segment and source of the secondary amino nitrogen

The unusual amino acid hypusine is produced in a single protein of mammalian cells by a novel posttranslational event in which a lysine residue is conjugated with the four-carbon moiety from the polyamine spermidine to form an intermediate deoxyhypusine, and in which this intermediate is subsequently hydroxylated. Specifically isotopically labeled precursors of hypusine were used to identify the biosynthetic origin of some of the atoms of hypusine and thus to provide further insight into the mechanism of this in vivo chemical modification reaction. Radiolabel from [1,4-3H] putrescine, [1,8-3H]spermidine, and [5-3H]spermidine entered hypusine during growth of Chinese hamster ovary cells. The occurrence of this label at positions 1 and 4, at position 4, and at position 1, respectively, in the 4-amino-2-hydroxybutyl portion of hypusine revealed an alignment of atoms identical to that in the butylamine segment of spermidine. Growth of cells with [epsilon-15N]lysine as the source of lysine yielded hypusine enriched in 15N, whereas only isotope-free hypusine during growth by [4-15N]spermidine. These was found in cells whose spermidine was replaced during growth by [4-15N]spermidine. These findings are in accordance with a proposal that the first phase of hypusine biosynthesis, the production of intermediate deoxyhypusine, occurs through transfer of the butylamine moiety from spermidine to the epsilon-amino nitrogen of protein-bound lysine. The technique of thermospray high-performance liquid chromatography/mass spectrometry provided positive identification of 15N in hypusine through final separation and on-column direct analysis of this amino acid. Methods of preparation are given for spermidine of high specific radioactivity, labeled specifically at position 5 with 3H, and for spermidine with 15N at the 4-position.     

Kappa-receptor selective binding of opioid ligands with a heterocyclic bicyclo[3.3.1]nonan-9-one structure

Previous pharmacological results have suggested that members of the heterocyclic bicyclo[3.3.1]nonan-9-one-like compounds are potent kappa-opioid receptor specific agonists. One lead molecule of this series. called compound 1 (dimethyl 7-methyl-2,4-di-2-pyridyl-3.7-diazabicyclo[3.3.1]nonan-9-one-1,5-dicarboxylate) exhibited high affinity for [3H]ethylketocyclazocine and [3H]U-69.593 binding sites in guinea pig cerebellar membranes which known to be a good source for kappa1 receptors. It was shown by molecular modelling that heterocyclic bicyclo[3.3.1]nonan-9-ones fit very well with the structure of ketazocine, a prototypic kappa-selective benzomorphan compound; when compared to the arylacetamide structure of U-69.593, a specific kappa1-receptor agonist, a similar geometry was found with a slightly different distribution of the charges. It is postulated, that the essential structural skeleton involved in the opioid activity is an aryl-propyl-amine element distributed along the N7-C6-C5-C4-aryl bonds.     

N-3(9)-arylpropenyl-N-9(3)-propionyl-3,9-diazabicyclo[3.3.1]nonanes as mu-opioid receptor agonists. Effects on mu-affinity of arylalkenyl chain modifications

Two series of N-3-arylpropenyl-N-9-propionyl-3,9-diazabicyclo[3.3.1]nonanes (1b-j) and of the reverted N-3-propionyl-N-9-arylpropenyl isomers (2b-j) as analogues of the previously reported analgesic N-3(9)-cinnamyl-N-9(3)-propionyl-3,9-diazabicyclo[3.3.1]nonanes (DBN) (1a, 2a) were synthesised and their affinity and selectivity towards opioid mu-, delta- and kappa-receptors were evaluated. Several compounds (1e,i,j-2d,e,f,g,j) exhibited a mu-affinity in the low nanomolar range with moderate or negligible affinity towards delta- and kappa-receptors. The representative term N-9-(3,3-diphenylprop-2-enyl)-N-3-propionyl-DBN (2d) displayed in vivo (mouse) a potent analgesic effect (ED(50) 3.88 mg/kg ip) which favourably compared with that of morphine (ED(50) 5 mg/kg ip). In addition, 2d produced in mice tolerance after a period twice as long with morphine.     

Selective modification of the catalytic subunit of cAMP-dependent protein kinase with sulfhydryl-specific fluorescent probes

The catalytic subunit of cAMP-dependent protein kinase contains only two cysteine residues, and the side chains of both Cys 199 and Cys 343 are accessible. Modification of the catalytic subunit by a variety of sulfhydryl-specific reagents leads to the loss of enzymatic activity. The differential reactivity of the two sulfhydryl groups at pH 6.5 has been utilized to selectively modify each cysteine with the following fluorescent probes: 3,6,7-trimethyl-4-(bromomethyl)-1,5-diazabicyclo[3.3.0]octa-3,6-diene- 2,8-dione, N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine, and 4-[N-[(iodoacetoxy)ethyl]-N-methyl-amino]-7-nitrobenz-2-oxa-1,3-diazole. The most reactive cysteine is Cys 199, and exclusive modification of this residue was achieved with each reagent at pH 6.5. Modification of Cys 343 required reversible blocking of Cys 199 with 5,5'-dithiobis(2-nitrobenzoic acid) followed by reaction of Cys 343 with the fluorescent probe at pH 8.3. Treatment of this modified catalytic subunit with reducing reagent restored catalytic activity by unblocking Cys 199. In contrast, catalytic subunit that was selectively labeled at Cys 199 by the fluorescent probes was catalytically inactive. Even though Cys 199 is presumably close to the interaction site between the regulatory subunit and the catalytic subunit, all of the modified C-subunits retained the capacity to aggregate with the type II regulatory subunit in the absence of cAMP, and the resulting holoenzymes were dissociated in the presence of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neomycin Is an Agonist at a Polyamine Site on the N-Methyl-D-Aspartate Receptor

Neomycin appears as a full agonist and spermidine as a partial agonist at the site where polyamines enhance 1-[1-(2-thienyl)cyclohexyl][3H]piperidine ([3H]TCP) binding on the N-methyl-D-aspartate (NMDA) receptor. Other aminoglycosides also enhance [3H]TCP binding with efficacies roughly proportional to the number of primary amine groups. The polyamine antagonists ifenprodil and arcaine inhibit enhancement of [3H]TCP binding by spermidine or neomycin. The inhibition of [3H]TCP binding by arcaine is apparently competitively reduced by neomycin and spermidine, supporting a common site. Diethylenetriamine (previously described as a polyamine antagonist) may be a partial agonist. Enhancement by neomycin or spermidine is not additive to that of Mg2+, consistent with competition of Mg2+ and spermidine or neomycin at the site where these compounds enhance [3H]TCP binding. Polyamines also enhance the binding of the competitive antagonist 2-(2-carboxypiperazin-4-yl)[3H]propyl-1-phosphonic acid ([3H]CPP). Neomycin, which does not enhance [3H]CPP binding, inhibits the enhancement by spermidine. That this site is distinct from the site where spermidine and neomycin increase [3H]TCP binding is supported by different pharmacology. Arcaine and diethylenetriamine do not inhibit spermidine enhancement of [3H]CPP binding. Mg2+ also does not compete with the spermidine enhancement of [3H]CPP binding. Ifenprodil inhibits the spermidine enhancement of [3H]CPP binding. The data suggest two or more polyamine sites, with arcaine selective for the site that enhances [3H]TCP binding. Neomycin is an agonist at one polyamine site and antagonist to the second.     

Toward the development of metal-free synthetic nucleases: cleavage of a model substrates by 1,4-diazabicyclo[2.2.2]octane derivatives

Artificial ribonucleases of A(n)BCL series were synthesized by solid-phase method. They consist of a hydrophobic alkyl radical A (n = 3-12 carbon atoms), an "RNA-binding domain" B (bisquaternary salt of 1,4-diazabicyclo[2.2.2]octane), a "catalytic domain" C (histidine residue) and a "linker" L that joins the domains B and C. The effect of the alkyl radical on the catalytic properties of the chemical catalyst was studied using three activated phosphate ester substrates: p-nitrophenyl phosphate, bis-p-nitrophenyl phosphate, and thymidine-3'-p-nitrophenyl phosphate.     

Investigations of the oxidative disassembly of Fe-S clusters in Clostridium pasteurianum 8Fe ferredoxin using pulsed-protein-film voltammetry

Rapid responses of biological [4Fe-4S] clusters to conditions of oxidative stress have been studied by protein-film voltammetry by using precise pulses of electrode potential to trigger reactions. Investigations with Clostridium pasteurianum 8Fe ferredoxin exploit the fact that [3Fe-4S] clusters display a characteristic pattern of voltammetric signals, so that their appearance and disappearance after an oxidative pulse can be tracked unambiguously under electrochemical control. Adsorbed to monolayer coverage at a graphite electrode, the protein initially shows a strong signal (B') at -0.36 V vs standard hydrogen electrode due to two [4Fe-4S](2+/+) clusters at similar potentials. Short square pulses (0.1-5 s) to potentials in the range 0.5-0.9 V cause extensive loss of B', and new signals appear (A'and C') that arise from [3Fe-4S] species (+/0 and 0/2- couples). The A' and B' intensities quantify transformations which are induced by the pulse and which occur subsequently when more reducing conditions are restored. Optimal [3Fe-4S] formation (in excess over [4Fe-4S]) is achieved with a 3-s pulse to 0.7 V, following which there is rapid partial recovery to yield a 1:1 3Fe:4Fe ratio, consistent with 7Fe protein. Thus, a 6Fe protein is formed, but one of the clusters is rapidly repaired. The [3Fe-4S]:[4Fe-4S] ratio follows a bell-shaped curve spanning the same potential range that defines complete loss of signals, while double-pulse experiments show that [3Fe-4S](+) resists further oxidative damage. Oxidative disassembly involves successive one-electron oxidations of [4Fe-4S] (i.e., 2+ --> 3+ --> 4+), with [3Fe-4S](+) being a relatively stable byproduct, that is, not an intermediate. Disassembly of [3Fe-4S] in the 7Fe protein continues after reducing conditions are restored, with lifetimes depending on oxidation level; thus 1+ (most stable) > 0 > 2-. In the presence of Fe(2+), the 0 level is stabilized by conversion back to [4Fe-4S](2+/+). By pulsing in the presence of Zn(2+), the [3Fe-4S] clusters that are formed are trapped rapidly as their Zn adducts.     

Imino [4+4] cycloaddition products as exclusive and biologically relevant acrolein-amine conjugates are intermediates of 3-formyl-3,4-dehydropiperidine (FDP), an acrolein biomarker

We demonstrated synthetically that the eight-membered heterocycles 2,6,9-triazabicyclo[3.3.1]nonanes and 1,5-diazacyclooctanes are the initial and exclusive products of the reaction, through an imino [4+4] cycloaddition, of biologically relevant amines with acrolein. The stabilities of the aminoacetals within the eight-membered heterocycles determined whether the product was subsequently transformed gradually into the 3-formyl-3,4-dehydropiperidine (FDP), which is widely used as an oxidative stress marker. The reactivity profiles discovered in this study suggested that some of the imino [4+4] cycloaddition products are reactive intermediates of FDP and contribute to the mechanisms underlying the oxidative stress response to acrolein.     

Oxidative cyclization of 2',3'-O-isopropylideneadenosines to 5'-O,8-cycloadenosines: considerations of N6-substituent effects and mechanism.

Oxidative cyclization of 2',3'-O-isopropylideneadenosines to the corresponding 5'-O,8-cyclo-2',3'-O-isopropylideneadenosines was achieved by using by lead tetraacetate, cupric chloride, and N-halogeno-succinimide as an oxidant, and by irradiation with a uv-visible light in the presence of pyrimido[5,4-g]pteridinetetrone 5-oxide. The reaction modes for the oxidative cyclization were investigated and discussed. In particular, N6-substituent effects on the oxidative cyclization provided a positive proof supporting the respective reaction mode.     

Two isoforms of eIF-5A in chick embryo. Isolation, activity, and comparison of sequences of the hypusine-containing proteins.

Eukaryotic translation initiation factor 5A (eIF-5A) (older terminology, eIF-4D) is unique in that it contains the unusual amino acid hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine). Hypusine is formed by a post-translational event in which a specific lysine residue is modified by a structural contribution from spermidine. Metabolic labeling of chick embryo fibroblasts with [3H]spermidine or [3H]lysine gives rise to two distinct proteins, designated I (approximately 20 kDa and pI 5.6) and II (approximately 18 kDa and pI 5.35), that contain [3H]hypusine. Upon incubation with [3H]lysine the labeling of the two proteins followed a similar time course and showed approximately the same ratio over the 6-h incubation period. [3H]Hypusine-containing proteins from cells which had been cultured with [3H]spermidine were employed as tracers for isolation of hypusine-containing proteins from whole chick embryos. Four such proteins were obtained. Two of these proteins, I and II, correspond to the two native proteins synthesized in chick embryo fibroblasts; the other two forms, Ia and IIa, displayed properties suggesting that they were derived from the native proteins, I and II, respectively, during purification. The amino acid compositions and the tryptic peptide maps of the 20-kDa protein (I) and the 18 kDa protein (II) suggest that they are closely related but distinct proteins. In fact, amino acid sequence analysis of the two major proteins revealed differences in the polypeptide backbone of the two proteins. In spite of structural differences, the two native forms (I and II), as well as the two altered forms (Ia and IIa), were effective in stimulating methionyl-puromycin synthesis, providing evidence that they are indeed functional isoforms of eIF-5A.     

Synthesis and pharmacological evaluation of bicyclic SNC80 analogues with separated benzhydryl moiety

Directed by molecular modeling studies the pharmacophoric benzhydryl moiety of the delta opioid receptor agonist SNC80 was separated and the two phenyl residues were attached to different positions of the conformationally constrained 6,8-diazabicyclo[3.2.2]nonane framework in order to find novel delta agonists. The crucial reaction step in the chiral pool synthesis was the establishment of the three carbon bridge by a Dieckmann analogous cyclization of the allyl and propyl derivatives 6 and 7 to yield the mixed methyl silyl acetals 8 and 9, respectively. Stereoselective Grignard reaction, dehydration, and introduction of the pharmacophoric (N,N-diethylcarbamoylbenzyl) residue led to the designed delta receptor agonists 3, ent-3, and 20 with a double bond in the bicyclic framework. Hydrogenation of the allyl derivative 14 was performed with ammonium formate and Pd/C to yield the saturated ligands 24a and 24b. Removal of the allyl substituent with RhCl(3), hydrogenation of the ring system, and re-attachment of the allyl moiety provided the allyl derivatives 4a and 4b. In receptor binding studies with the radioligand [(3)H]-deltorphine II only ent-3 showed considerable delta receptor affinity (K(i)=740 nM). Since ent-3 also interacts with mu receptors (K(i)=250 nM) it belongs to the very interesting compound class of mixed delta/mu ligands.     

Cooperative Modulation of [3H]MK-801 Binding to the N-Methyl-d-Aspartate Receptor-Ion Channel Complex by l-Glutamate, Glycine, and Polyamines

In extensively washed rat cortical membranes [3H](+)-5-methyl-10,11-dihydro-5 H-dibenzo [a,d]cyclohepten-5,10-imine ([3H]MK-801) labeled a homogeneous set of sites (Bmax = 1.86 pmol/mg protein) with relatively low affinity (KD = 45 nM). L-Glutamate, glycine, and spermidine produced concentration-dependent increases in specific [3H]MK-801 binding due to a reduction in the KD of the radioligand. In the presence of high concentrations of L-glutamate, glycine, or spermidine, the KD values for [3H]MK-801 were reduced to 11 nM, 18 nM, and 15 nM, respectively. Maximally effective concentrations of combinations of the three compounds further increased [3H]MK-801 binding affinity as follows: L-glutamate + glycine, KD = 6.2 nM; L-glutamate + spermidine, KD = 2.2 nM; glycine + spermidine, KD = 8.3 nM. High concentrations of spermidine did not inhibit either [3H]glycine orf [3H]3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid binding to the N-methyl-D-aspartate (NMDA) receptor complex. The concentration of L-glutamate required to produce half-maximal enhancement (EC50) of [3H]MK-801 binding was reduced from 218 nM to 52 nM in the presence of 30 microM glycine and to 41 nM in the presence of 50 microM spermidine. The EC50 value for glycine enhancement of [3H]MK-801 binding was 184 nM. This was lowered to 47 nM in the presence of L-glutamate and to 59 nM in the presence of spermidine. Spermidine enhanced [3H]MK-801 binding with an EC50 value of 19.4 microM which was significantly reduced by high concentrations of L-glutamate (EC50 = 3.9 microM) or glycine (EC50 = 6.2 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

The identification of an eukaryotic initiation factor 4D precursor in spermidine-depleted Chinese hamster ovary cells

When Chinese hamster ovary cells are incubated with [terminal methylenes-3H]spermidine, radioactivity is incorporated into a single cellular protein, eukaryotic initiation factor 4D (eIF-4D), through posttranslational synthesis of the amino acid hypusine (N epsilon-(4-amino-2-hydroxybuyly)lysine). The effect of spermidine depletion on this protein modification reaction was studied by high resolution two-dimensional gel electrophoresis. Factor eIF-4D containing both [3H]lysine and [3H]hypusine was detected as one of the major labeled cellular proteins on the fluorographic map of the proteins from Chinese hamster ovary cells that had been incubated with [3H]lysine. When these cells were depleted of spermidine by the use of DL-alpha-difluoromethylornithine before addition of [3H]lysine, no radiolabeling of this mature eIF-4D (hypusine form, Mr approximately 18,000; pI approximately 5.3) occurred. Instead, a new radiolabeled protein (Mr 18,000; pI 5.1) that contained [3H]lysine but no [3H]hypusine or [3H]deoxyhypusine was seen. This protein was identified as an eIF-4D precursor by comparison of the two-dimensional map of its tryptic peptides with that of the tryptic peptides from [3H]lysine-labeled eIF-4D. Further comparisons also suggest that additional post-translational modification processes are involved in the biogenesis of eIF-4D.     

Cyclization under mild conditions of salicyloyl-dipeptides

Carboxy-activated linear peptides 6(a-c) of general formula Sal-Xaa-Pro-ONp (Xaa = Phe: Gly; Aib) were synthesized and treated at room temperature with 1,8-diazabicyclo [5,4,0] undec-7-ene (DBU) in benzene solution. The tetrahedral adducts (oxa-cyclols) 7, 11 and 12, tautomeric forms of the corresponding 10-membered cyclodepsitripeptides, have been isolated in each of the three models examined. These adducts, which contain the hydroxylated carbon atom located at the junction between two 6-membered rings, do not show a tendency to isomerize into the corresponding macrocyclic lactones, regardless of the nature of the substituents on the C alpha carbon atom of the central residue. Partially cyclized dimeric products 8 and 13, identified as N-(Sal-Xaa-Pro)-dioxopiperazines (Xaa = Phe;Aib), have been also isolated from the cyclization reactions.     

Characterization of Two Cerebellar Binding Sites of[3H]Ro 15-4513

Ro 15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H- imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate), a partial inverse agonist of central benzodiazepine receptors, binds to two distinct sites in the cerebellum. The binding to diazepam-sensitive (DZ-S) sites is displaced by different benzodiazepine receptor ligands, whereas the other site is insensitive to benzodiazepine agonists [diazepam-insensitive (DZ-IS)]. The binding of [3H]Ro 15-4513 was studied in pig cerebellar membranes and in receptors solubilized and purified from these. Micromolar concentrations of gamma-aminobutyric acid (GABA) decreased DZ-S binding at both 0 and 37 degrees C, whereas it had no effect on DZ-IS binding at 0 degrees C and was stimulatory at 37 degrees C. The pH profiles of [3H]Ro 15-4513 binding were quite similar in both binding sites in the pH range of 5.5-10.5 but differed at acidic pH values from those reported for flunitrazepam and Ro 15-1788 (flumazenil; ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H- imidazol[1,5-a][1,4]benzodiazepine-3-carboxylate) binding in DZ-S sites, suggesting that [3H]Ro 15-4513 does not interact with a histidine residue apparently present in the binding site. Zn2+, Cu2+, Co2+, and Ni2+ enhanced the binding to DZ-S sites, and the first three mentioned also enhanced the binding to DZ-IS sites. [3H]Ro 15-4513 binding activity was solubilized by various detergents. All detergents tested were more efficient in solubilizing DZ-S binding activity. High ionic strength improved especially the solubility of DZ-IS binding activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and content of polyamines in bloodstream Trypanosma brucei

The sensitive dansyl procedure was used to detect putrescine and spermidine, but not spermine and cadaverine, in pleomorphic Trypanosoma brucei. The polyamines were synthesized in vitro from [3H]ornithine, [14C]arginine and [14C]methionine. Proline, agmatine, and citrulline, but not glutamine, glutamic or pyroglutamic acids, stimulated spermidine formation from [4C]methionine. Putrescine and sperimidine synthesis occurred rapidly from ornithine: putrescine synthesis peaked in 0.5 h, spermidine in 1 h. Trypanosoma brucei assimilated exogenous 14C-labeled putrescine, spermidine, and spermine; spermidine and spermine were taken up 5 times as rapidly as putrescine. Polyamine syntheses may therefore be a practical target for novel trypanocies.     

Conformationally constrained ethylenediamines: synthesis and receptor binding of 6,8-diazabicyclo[3.2.2]nonanes

The synthesis and receptor affinity of 6,8-diazabicyclo[3.2.2]nonanes representing conformationally constrained ethylenediamines are described. The Dieckmann analogous cyclization of the (piperazin-2-yl)propionate 9 provided the bicyclononane 10 only, when the first cyclization product was trapped with chlorotrimethylsilane. 10 was stereoselectively transformed into the bicyclic amines 19a,b and amides 22a,b, which were investigated in competition experiments with radioligands for their sigma(1)-, sigma(2)-, kappa-, and mu-receptor affinities. The (2R)-configured dimethylamine 19a showed promising sigma(1)-receptor affinity (K(i)=23.8 nM) and selectivity, whereas the (2S)-configured (dichlorophenyl)acetamide 22b displayed a sigma-receptor binding profile (sigma(1): K(i)=184 nM; sigma(2): K(i)=263 nM) very similar to the binding profile of the atypical antipsychotic BMY-14802 (26).