Spermidine Analogues Research Articles

The efficacy of inhibitors involved in spermidine metabolism in Plasmodium falciparum, Anopheles stephensi and Trypanosoma evansi.

In the present study, we have tested the effect of different polyamine inhibitors of the spermidine metabolizing enzymes deoxyhypusine synthase and homospermidine synthase in different chloroquine resistant Plasmodium falciparum strains, in the mosquito Anopheles stephensi (Diptera: Culicidae) and in a Trypanosoma evansi clone I from strain STIB 806 K China. Recent experiments have shown that agmatine is a growth inhibitor of the malaria parasite P. falciparum (Kaiser et al. 2001) in vitro. A comparison of agmatine efficacy with the new antimalarials artemisinin, triclosan and conventional chloroquine showed similar or even better results on the basis of growth inhibition and the reduction of developmental forms. However, no effect of triclosan or agmatine was observed at the ribonucleic acid level. In a second set of experiments, we tested the effect of 1,7-diaminoheptane and agmatine on oocyst formation in A. stephensi after infection with Plasmodium yoelii. Agmatine had an antisporozoite effect since 1,000 microM led to a 59.5% inhibition of oocysts. A much weaker inhibitor of oocyst formation was 1,7-diaminoheptane. The most effective in in vitro inhibition of T. evansi was dicyclohexylamine, an inhibitor of spermidine biosynthesis with an IC(50 ) value of 47.44 microM and the deoxyhypusine inhibitor 1,7-diaminoheptane with an IC(50) value of 47.80 microM. However, both drugs were ineffective in in vivo experiments in a Trypanosoma mouse model. Two different spermidine analogues, 1,8-diaminooctane and 1,3-diaminopropane with IC(50) values of 171 microM and 181.37 microM, respectively, were moderate inhibitors in vitro and ineffective in vivo.

TATA-binding Protein-associated Factor 7 Regulates Polyamine Transport Activity and Polyamine Analog-induced Apoptosis

Identification of the polyamine transporter gene will be useful for modulating polyamine accumulation in cells and should be a good target for controlling cell proliferation. Polyamine transport activity in mammalian cells is critical for accumulation of the polyamine analog methylglyoxal bis(guanylhydrazone) (MGBG) that induces apoptosis, although a gene responsible for transport activity has not been identified. Using a retroviral gene trap screen, we generated MGBG-resistant Chinese hamster ovary (CHO) cells to identify genes involved in polyamine transport activity. One gene identified by the method encodes TATA-binding protein-associated factor 7 (TAF7), which functions not only as one of the TAFs, but also a coactivator for c-Jun. TAF7-deficient cells had decreased capacity for polyamine uptake (20% of CHO cells), decreased AP-1 activation, as well as resistance to MGBG-induced apoptosis. Stable expression of TAF7 in TAF7-deficient cells restored transport activity (55% of CHO cells), AP-1 gene transactivation (100% of CHO cells), and sensitivity to MGBG-induced apoptosis. Overexpression of TAF7 in CHO cells did not increase transport activity, suggesting that TAF7 may be involved in the maintenance of basal activity. c-Jun NH2-terminal kinase inhibitors blocked MGBG-induced apoptosis without alteration of polyamine transport. Decreased TAF7 expression, by RNA interference, in androgen-independent human prostate cancer LN-CaP104-R1 cells resulted in lower polyamine transport activity (25% of control) and resistance to MGBG-induced growth arrest. Taken together, these results reveal a physiological function of TAF7 as a basal regulator for mammalian polyamine transport activity and MGBG-induced apoptosis.

Characterization by Raman spectroscopy of conformational changes on guanine–cytosine and adenine–thymine oligonucleotides induced by aminooxy analogues of spermidine

AbstractThe interactions between aminooxy analogues of the biogenic polyamine spermidine and selected 15‐mer oligodeoxyribonucleotides with alternating purine–pyrimidine base sequences, adenine–thymine (AT) and guanine–cytosine (GC), have been studied using Raman spectroscopy. These analogues resulted from the substitution of the two terminal aminomethylene groups of spermidine, +NH3CH2, by an aminooxy one, H2NO. Since this biogenic polyamine has no centre of symmetry, there are two different isosteric molecules, which have been named AOEPUT and APAPA. However, the pKa of an aminooxy group (about 5) is not high enough to be protonated at neutral pH. Therefore, under physiological conditions, these analogues have a positive charge less than spermidine, which makes them suitable models to investigate the roles of both charge and structure in the polyamine–DNA interactions. Raman spectra of solutions containing different concentrations of the analogues, from 10 to 75 mM, and either a GC and an AT oligonucleotide, at a fixed concentration 60 mM (in phosphate), were recorded. The spectra demonstrated the existence of strong differences in the oligonucleotide‐analogue interactions depending on the base sequences. In the presence of the aminooxy analogues, a 15‐mer GC oligonucleotide adopted a highly ordered tertiary structure (aggregate state or Ψ‐DNA) and, at the same time, it changed its secondary structure (from B to Z‐DNA). Evidence of macromolecular transitions was not achieved from the Raman spectra of a 15‐mer AT oligonucleotide. However, the assignments of the Raman bands indicated that both H2NO groups of the aminooxy analogues and exocyclic NH2 groups of the oligonucleotides could act as preferential binding sites. However, different spectral features were observed between AOEPUT and APAPA in the presence of the oligonucleotides. This fact supports the idea that the two amino terminal groups of spermidine could have different roles in the interaction of this biogenic polyamine with DNA. Our results also confirm that the aminooxy analogues of biogenic polyamines could act as stabilizers of non‐viral transfection vectors, thus improving the efficiency of gene therapy protocols. Copyright © 2004 John Wiley & Sons, Ltd.

Recent Advances in the Synthesis of Spermine and Spermidine Analogues of the Shark Aminosterol Squalamine

AbstractFor Abstract see ChemInform Abstract in Full Text.

Solution aggregation of anti-trypanosomal N-(2-naphthylmethyl)ated polyamines

Trypanosomatidae parasites are responsible for many human and animal diseases including African sleeping sickness, Chagas’ disease, and Nagana cattle disease. Since current treatment of trypanosome infections is difficult and often ineffective in controlling the chronic phases of these diseases, more effective anti-trypanosomal drugs are urgently needed. One class of polyamines containing hydrophobic side chains shows promise. However, conformational information regarding their interaction with the target enzyme trypanothione reductase has yet to be obtained. As prelude to such studies, we have made preliminary studies of novel spermine and spermidine analogs bearing one or two N-substituted 2-naphthylmethyl groups dissolved in aqueous solution. Our studies suggest the pH-dependent formation of fluorescent aggregates involving either the encounter of two excited-state naphthyl groups (“excimer”) or formation of an excited-state complex (“exciplex”) formed as a consequence of amine-to-naphthyl electron transfer. These spectral changes may be used to explore the mechanism by which N-(2-naphthylmethyl) polyamine analogs exert their toxic effects toward the design of improved candidates for anti-trypanosomal chemotherapy.

Recent Advances in the Synthesis of Spermine and Spermidine Analogs of the Shark Aminosterol Squalamine

AbstractGreat attention has recently been focused on the shark aminosterol squalamine (1) because of its important antimicrobial and antiangiogenic activities. Herein, we present the different series of spermine and spermidine analogs that have been synthesized. Many of the squalamine mimics demonstrate a broad spectrum activity against microorganisms. Squalamine is currently undergoing Phase II clinical trials in cancer patients. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)

Cover Picture: Recent Advances in the Synthesis of Spermine and Spermidine Analogs of the Shark Aminosterol Squalamine (Eur. J. Org. Chem. 20/2003)

AbstractThe cover picture shows, the dogfish shark Squalus Acanthias from which the aminosterol squalamine has been isolated. Squalamine has been characterized as a water‐soluble cationic steroid exhibiting potent antimicrobial activity against fungi, protozoa fungi, protozoa and both Gram‐negative and Gram‐positive bacteria. Details on the different series of spermine and spermidine analogs synthesized as well as comparison of their pharmacological properties are discussed in the Microreview by J. M. Brunel and Y. Letourneux on p. 3897 ff.

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.

A Polyamine Analogue Prevents Acute Pancreatitis and Restores Early Liver Regeneration in Transgenic Rats with Activated Polyamine Catabolism

We recently generated a transgenic rat model for acute pancreatitis, which was apparently caused by a massive depletion of pancreatic polyamines spermidine and spermine due to inducible activation of their catabolism (Alhonen, L., Parkkinen, J. J., Keinänen, T., Sinervirta, R., Herzig, K. H., and Jänne, J. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 8290-8295). When subjected to partial hepatectomy, these animals showed striking activation of polyamine catabolism at 24 h postoperatively with a profound decrease in hepatic spermidine and spermine pools and failure to initiate liver regeneration. Here we show that pancreatitis in this model could be totally prevented, as judged by histopathology and plasma alpha-amylase activity, by administration of 1-methylspermidine, a metabolically stable analogue of spermidine. Similarly, the analogue, given prior to partial hepatectomy, restored early liver regeneration in the transgenic rats, as indicated by a dramatic increase in the number of proliferating cell nuclear antigen-positive hepatocytes from about 1% to more than 40% in response to the drug. The present results suggest that the extremely high concentration of spermidine in the pancreas, in fact the highest in the mammalian body, may have a critical role in maintaining organ integrity. The failure to initiate liver regeneration in the absence of sufficient hepatic polyamine pools similarly indicates that polyamines are required for proper commencement of the regenerative process.

(Z)-1,4-Diamino-2-butene as a vector of boron, fluorine, or iodine for cancer therapy and imaging: synthesis and biological evaluation

Polyamine vectors are attractive for tumor targeting. We envisaged (Z)-1,4-diamino-2-butene ( Z-DAB ), an unsaturated analogue of putrescine as vector of 10B, 18F and 131I for boron neutron capture therapy (BNCT), and tumor imaging by positron emission tomography or scintigraphy respectively. In the present work, the synthesis and characterization of new derivatives of Z-DAB were reported. Z-DAB was actively transported in cells via the polyamine transport system and converted into the spermidine analogue. (E)-2-iodo-1,4-diamino-2-butene ( E-I-DAB ) was not taken up by the polyamine transport system and may not be suitable for tumor imaging. In contrast, (Z)-2-{4-(5,5-dimethyl- [1,3,2]dioxaborinan-2-yl)phenyl}methyl-1,4-diamino-2-butene ( Z-4-Bbz-DAB ) was a substrate of the transport system and allowed significant boron accumulation in 3LL cells. Its potential in BNCT will be evaluated.

Synthesis of six novel N, N-dialkyl derivatives of spermidine and effects on growth of the fungal plant pathogen Pyrenophora avenae

Six novel N, N-dialkyl derivatives of spermidine were synthesised and examined for activity against the oat stripe pathogen Pyrenophora avenae. Two of these spermidine analogues, N, N-dimethyl- N 1-(3-aminopropyl)-1,3-diaminopropane trihydrochloride ( 27) and N, N-dimethyl- N 1-(3-aminopropyl)-1,4-diaminobutane trihydrochloride ( 28), reduced radial extension of P. avenae on plates when used at 2 mM, and caused more substantial reductions in fungal growth in liquid culture when used at 1 mM. Preliminary data suggest that neither compound affected polyamine biosynthesis, determined by following the incorporation of label from ornithine into polyamines and examining intracellular polyamine concentrations in fungal tissue.

Synthesis of six novel N,N-dialkyl derivatives of spermidine and effects on growth of the fungal plant pathogen Pyrenophora avenae.

Six novel N,N-dialkyl derivatives of spermidine were synthesised and examined for activity against the oat stripe pathogen Pyrenophora avenae. Two of these spermidine analogues, N,N-dimethyl-N1-(3-aminopropyl)-1,3-diaminopropane trihydrochloride (27) and N,N-dimethyl-N1-(3-aminopropyl)-1,4-diaminobutane trihydrochloride (28), reduced radial extension of P. avenae on plates when used at 2 mM, and caused more substantial reductions in fungal growth in liquid culture when used at 1 mM. Preliminary data suggest that neither compound affected polyamine biosynthesis, determined by following the incorporation of label from ornithine into polyamines and examining intracellular polyamine concentrations in fungal tissue.

Interaction of DNA with an aminooxy analogue of spermidine — an FT-IR and FT-Raman approach

The interaction of highly polymerized calf-thymus DNA with 1-aminooxy-3-N-(3-aminopropyl)-aminopropane (ap-apa), an aminooxy analogue of the biogenic ornithine-derived polyamine spermidine, has been investigated by vibrational spectroscopy. Infrared and Raman spectra of DNA/ap-apa solutions, at different polyamine concentrations, were registered. The infrared spectra were extended to solutions in heavy water in order to analyze the 1500–1700cm−1 region. The spectroscopical data were discussed in terms of preferential binding sites between DNA and the aminooxy polyamine. The results support the existence of structural specifities in the interaction, at least under our experimental conditions; they also indicate differences at low and high ap-apa concentrations, which involve mainly base residues.

Guanidino-containing drugs in cancer chemotherapy: biochemical and clinical pharmacology

The pharmacology and clinical application of three guanidino-containing compounds are reviewed in this commentary with special focus on a new member of this group of drugs, CHS 828 [ N-(6-(4-chlorophenoxy)hexyl)- N′-cyano- N″-4-pyridylguanidine]. m-Iodobenzylguanidine (MIBG) and methylglyoxal bis(guanylhydrazone) (MGBG) have been extensively studied, preclinically as well as clinically, and have established use as anticancer agents. MIBG has structural similarities to the neurotransmitter, norepinephrine, and MGBG is a structural analog of the natural polyamine spermidine. CHS 828 is a pyridyl cyanoguanidine newly recognized as having cytotoxic effects when screening antihypertensive compounds. Apart from having the guanidino groups in common, there are many differences between these drugs in both structure and their mechanisms of action. However, they all inhibit mitochondrial function, a seemingly unique feature among chemotherapeutic drugs. In vitro in various cell lines and primary cultures of patient tumor cells and in vivo in various tumor models, CHS 828 has cytotoxic properties unlike any of the standard cytotoxic drugs with which it has been compared. Among these are non-cross-resistance to standard drugs and pronounced activity in tumor models acknowledged to be highly drug-resistant. Similar to MIBG, CHS 828 induces an early increase in extracellular acidification, due to stimulation of the glycolytic flux. Furthermore, ATP levels decrease, and the syntheses of DNA and protein are shut off after approximately 30 hr of exposure, indicating active cell death. CHS 828 is now in early clinical trials, the results of which are eagerly awaited.

Human deoxyhypusine synthase: interrelationship between binding of NAD and substrates

Deoxyhypusine synthase catalyses the NAD-dependent transfer of the butylamine moiety from the polyamine, spermidine, to a specific lysine residue of a single cellular protein, eukaryotic translation-initiation factor 5A (eIF5A) precursor. The native enzyme exists as a tetramer of four identical subunits and contains four binding sites for NAD. The binding of spermidine and NAD was studied by a filtration assay. [(3)H]Spermidine binding to the enzyme was not detectable alone or in the presence of the eIF5A precursor, but was detected only in the presence of NAD or NADH, suggesting that a NAD/NADH-induced conformational change is required for the binding of spermidine. A strong NAD-dependent binding was also observed with a spermidine analogue, N(1)-guanyl-1, 7-diamino[(3)H]heptane (GC(7)), but not with [(14)C]putrescine or [(14)C]spermine. Although [(3)H]NAD binding to the enzyme occurred in the absence of spermidine, its affinity for the enzyme was markedly enhanced by spermidine, GC(7) and also by the eIF5A precursor. The maximum binding for NAD and spermidine was estimated to be approximately 4 molecules each/enzyme tetramer. The dependence of spermidine binding on NAD and the modulation of binding of NAD by spermidine and the eIF5A precursor suggest intricate relationships between the binding of cofactor and the substrates, and provide new insights into the reaction mechanism.

Novel aminooxy analogues of spermidine and spermine impair polyamine uptake in Caco-2 cells

Novel aminooxy analogues of spermidine and spermine impair polyamine uptake in Caco-2 cells

SYNTHÈSE DE PHOSPHINATES ANALOGUES STRUCTURAUX PHOSPHORÉS DE LA PUTRESCINE ET DE LA SPERMIDINE

The phosphinates 1 and 2, phosphorylated analogues of putrescine and spermidine. were synthesized from a common precursor. ethyl(phtalimidomethyl)vinylphosphinate (9) by monochlorination of diethyl(phtalimidomethyl)phosphonate (7) and subsequent treatment with vinylmagnesium bromide. Michael addition of benzylamine or 3-azidopropylamine to 9 followed by catalytic hydrogenation (Pd/C) afforded 1 and 2 in good overall yields.

Interactions of polyamines with the DNA octamers d(m5CG)4 and d(GGAATTCC): A1H-NMR investigation

The interaction of two DNA octamers, d(m5CG)4 and d(GGAATTCC), with the polyamines spermine4+ and spermidine3+, has been studied by means of 1H-nmr nuclear Overhauser effect (NOE) difference measurements. The experiments were performed at 10°C and for a polyamine charge to DNA charge (i.e., phosphate) ratio of 0.4, where the solution of d(m5CG)4 contains about 50% Z-form of the DNA. The results show that the polyamine intramolecular NOEs for the protons on the propyl chains are similarly negative with the two oligonucleotides, while those on the butyl chain show slightly more negative NOE with d(m5CG)4 than with d(GGAATTCC). The fully N-methylated analogues of spermine (Me10Spn4+) and spermidine (Me8Spd3+) as well as the diamines 1,3-diaminopropane (DAP2+) and 1,4-diaminobutane (putrescine2+) have been studied for the ability to transform d(m5CG)4 from the B- to the Z-form. 1H-nmr spectra showed the order spermine4+ > spermidine3+ > Me10Spn4+ > Me8Spd3+ > 1,3-diaminopropane2+ > putrescine2+, with spermine showing the largest relative amount of Z-DNA. 1H-nmr pulsed-gradient self-diffusion measurements of the triamines showed a large difference in the interaction of Spd and Me8Spd with the two different duplexes. With the same duplex (either of the two), however, no difference between Spd and Me8Spd can be seen. Within a two-state model this is interpreted as a larger fraction of bound polyamines with d(m5CG)4 than with d(GGAATTCC). © 1999 John Wiley & Sons, Inc. Biopoly 49: 41–53, 1999

The effect of acylated polyamine derivatives on polyamine uptake mechanism, cell growth, and polyamine pools in Escherichia coli, and the pursuit of structure/activity relationships

Two acetyl analogues of spermidine and five analogues of spermine were used to determine the structural specificity of the polyamine transport system in Escherichia coli by measuring their ability to compete with [14C]putrescine or [14C]spermine for uptake, as well as to inhibit cell growth, and, finally, to affect the intracellular polyamine pools. Spermine uptake follows simple Michaelis-Menten kinetics (Kt = 24.58 +/- 2.24 microM). In contrast, the putrescine uptake system involves two saturable Michaelis-Menten carriers exhibiting different affinity towards putrescine (Kt = 3.63 +/- 0.43 microM, Kt' = 0.61 +/- 0.10 microM). From the Ki values, it is inferred that N1-5-amino-2-nitrobenzoylspermine is the most effective competitive inhibitor followed by N1-acetylspermine, and then N1,N12-diacetylspermine. N1-acetylspermidine and N8-acetylspermidine also inhibit competitively the uptake of spermine, the latter being the most effective inhibitor. In addition, the above-mentioned analogues inhibit identically one of the carriers of putrescine uptake, suggesting the existence of a common transporter for both putrescine and spermine. The order of analogue potency, regarding the other carrier of putrescine is as follows: N1,N12-diacetylspermine approximately N1-5-amino-2-nitro-benzoylspermine > N1-acetylspermine. Both N1-acetylspermidine (Ki = 753 +/- 25 microM, Ki' = 128 +/- 5 microM) and N8-acetylspermidine (Ki = 22.4 +/- 0.4 microM, Ki' = 279 +/- 3 microM) also cause competitive inhibition of putrescine uptake, however with inverse affinity towards the putrescine carriers. Neither N4,N9-diacetylspermine, nor N1,N4-bis(beta-alanyl)diaminobutane affect the uptake of any polyamine. Interestingly, none of the acetyl analogues of spermine has a measurable effect on cell growth and cellular polyamine pools, although some of them are accumulated in cells. Based on these findings, the relative significance of the primary and secondary amines and of the chain flexibility as determinants of cellular uptake are discussed.

Effects of cyclohexylamine and norspermidine on powdery mildew infection of spring barley

The spermidine synthase inhibitor cyclohexylamine (CHA) and the spermidine analogue norspermidine were evaluated against powdery mildew infection of spring barley in field trials over a 2-year period. Both compounds reduced mildew infection as effectively as a commercial fungicide, a mixture of flutriafol and carbendazim. Percentage disease reduction afforded by CHA and norspermidine in year 1 ranged from 32% to 67% at the final assessment and from 52% to 77% in year 2 of the study, when compared with plants from untreated plots. No difference was observed in the disease control conferred by 1.0 mM concentrations of the compounds and 5.0 mM applications, the optimum dose being less than 5.0 mM. No significant differences in final yield were obtained, comparing the treated crop with the untreated control. Although reductions in disease were apparent, additional environmental factors may have prevented a subsequent crop yield response.