Fluorinated analogues of spermidine as substrates of spermine synthase

Baillon, Jean; Mamont, Pierre S.; Wagner, Joseph; Gerhart, F.; Lux, Philippe

doi:10.1111/j.1432-1033.1988.tb14274.x

Cited by 29 publications

(22 citation statements)

References 20 publications

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“…The enzyme was purified to homogeneity by spermine-Sepharose affinity chromatography as previously described for the brain enzyme [IS]. Nucleic acid and polyamine-free HTC cell extract, prepared as previously described [12], was used for spermine synthase activity.…”

Section: Enzymesmentioning

confidence: 99%

Inhibition of mammalian spermine synthase by N‐alkylated‐1,3‐diaminopropane derivatives in vitro and in cultured rat hepatoma cells

Baillon¹,

Kolb²,

Mamont³

1989

European Journal of Biochemistry

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A number of N-alkylated-l,3-diaminopropane derivatives [H2N-(CH2)3-NH-(CH2),,H, where n = 1 -91 have been tested as potential inhibitors of partially purified rat hepatoma (HTC) cell or pure bovine spleen spermine synthase.Among the compounds described in this paper, the most potent competitive inhibitor of spermine synthase, with respect to spermidine, is N-butyl-l,3-didminopropane with Ki values of 11.9 nM and 10.4 nM for the HTC cell and bovine spleen enzymes respectively. Inhibition of spermine synthase by this alkylated amine is selective since spermidine synthase activity is not affected up to 100 pM N-butyl-1,3-diaminopropane at a range of 5 -200 pM putrescine.Added to the culture medium of growing HTC cells, N-butyl-l,3-diaminopropane causes the expected changes in the polyamine levels with a marked decrease of spermine and an increase of spermidine. Under these conditions cell growth continues unabated.Such N-alkylated-I ,3-diaminopropane derivatives may have considerable potential as tools for studying the role of polyamines and in particular the functions of spermine in cell multiplication and differentiation.The requirement of polyamine synthesis for eukaryotic cell multiplication has been demonstrated mainly by the use of specific irreversible inhibitors of L-ornithine decarboxylase, one of the key enzymes of the polyamine biosynthetic pathway (reviewed in [l]). Inhibition of L-ornithine decarboxylase has been shown to exert beneficial effects in animal models of proliferative diseases [2 -51 and in some protozoan infections in humans [6]. Altogether these studies emphasize the importance of the polyamine metabolic pathway as promising target for the design of new anti-proliferative agents [l, 21.Use of L-ornithine-decarboxylase inhibitors for creating polyamine deficiency in mammalian tissues has however certain limitations. In vivo administration of these compounds causes putrescine and spermidine deficiency but scarcely affects tissue spermine concentrations [7, 81. As hypothesized [9, lo], maintenance of residual cell growth and lack of cytotoxicity of these L-ornithine decarboxylase inhibitors may possibly result from their ineffectiveness in altering initial spermine levels. There is some experimental support for this hypothesis, at least in cultured cells [lo, I I]. However, availability of specific and very effective inhibitors of spermine synthase should provide more direct information on particular biological functions of spermine both in vitro and in vivo.Correspondence to P. S. Marnont, Merrell Dow Research Institute,

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Section: Enzymesmentioning

confidence: 99%

Inhibition of mammalian spermine synthase by N‐alkylated‐1,3‐diaminopropane derivatives in vitro and in cultured rat hepatoma cells

Baillon¹,

Kolb²,

Mamont³

1989

European Journal of Biochemistry

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“…1. The carbon atoms are numbered following Baillon et al [39]. Observed 13 C chemical shift profiles as a function of pD are illustrated in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The iterative process was not needed for spermidine (6) as the basicity constants used for the calculation of the enthalpies were based on published values [39] related to measurements at 25°C.…”

Section: Resultsmentioning

confidence: 99%

“…The superscipts a and b for the pairs of nuclei with very similar chemical shifts (C1, C12; C2, C11; and C3, C10) refer to the upper and lower lines, respectively the fact that we have re-determined the protonation enthalpies used to adjust the values to 25 °C, whereas before we used the enthalpies published by Palmer et al [41]. The basicity constants for 3 and 4 have been published by Baillon et al [39] from potentiometric data obtained at 25°C in the medium 0.1 M KNO 3 . Their values, log K 1 = 10.40, log K 2 = 9.55, log K 3 = 7.18, for (3), and log K 1 = 10.34, log K 2 = 9.29, log K 3 = 5.70, for (4) are in satisfactory agreement with the values obtained in the present work.…”

Section: Discussionmentioning

confidence: 96%

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Determination of protonation constants of some fluorinated polyamines by means of 13 C NMR data processed by the new computer program HypNMR2000. Protonation sequence in polyamines

Frassineti

Alderighi

Gans

et al. 2003

Analytical and Bioanalytical Chemistry

154

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The pK a values of 6-fluoro-4,8-diazadodecane-1,12-diamine (6-fluorospermine) (1), 6,6-difluoro-4,8-diazadodecane-1,12-diamine (6,6-difluorospermine) (2), 6-fluoro-4-azaoctane-1,8-diamine (6-fluorospermidine) (3) and 6,6-difluoro-4-azaoctane-1,8-diamine (6,6-difluorospermidine) (4) in D 2 O solution have been determined at 40°C from 13 C NMR chemical shifts data using the new computer program HypNMR2000. The enthalpies of protonation of compounds 1-4 and the parent amines spermine (5) and spermidine (6) have been determined from microcalorimetric titration data. The values of ∆H°were used to derive basicity constants relative to 25°C. The NMR data have been analysed by two different methods to obtain information on the protonation sequence in the polyamines 1-5. The protonation sequence for spermine is related to its biological activity.

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“…The proportion of the N 1 ,N 8 -diprotonated species of spermidine, which is the active amine substrate for this enzyme, is highest at this pH (31,32). The K m values for spermidine at pH 7.5 and pH 9.5 are 150 and 7 M, respectively (28,31 (31).…”

Section: Reversal Of Deoxyhypusine Synthesis Reaction and Identificatmentioning

confidence: 87%

Reversal of the Deoxyhypusine Synthesis Reaction

Park

Wolff

Folk

et al. 2003

Journal of Biological Chemistry

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Deoxyhypusine synthase catalyzes the first step in hypusine (N ⑀ -(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 3 H]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 [ 14 C]putrescine can replace eIF5A precursor protein as an acceptor of the 4-aminobutyl moiety of spermidine to form radiolabeled homospermidine. The K m value for putrescine (1.12 mM) as a 4-aminobutyl acceptor, however, is much higher than that for eIF5A precursor (1.5 M). Using [ 14 C]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 post-translational synthesis of an unusual amino acid, hypusine, converts the inactive eIF5A 1 precursor to an active protein (see Refs. 1-3). The biosynthesis of hypusine occurs exclusively in this protein by way of two enzymatic steps. In the first step, deoxyhypusine synthase (EC 2.5.1.46) facilitates the transfer of the 4-aminobutyl moiety from spermidine, the donor substrate, to the eIF5A precursor (eIF5A(Lys)), the acceptor substrate, to form a deoxyhypusine-containing protein (eIF5A(Dhp)) (4 -6). This intermediate is hydroxylated by deoxyhypusine hydroxylase (EC 1.14.99.29) (7) to complete hypusine synthesis and eIF5A maturation. The essential role of eIF5A and its hypusine modification is supported by gene disruption studies in yeast, Saccharomyces cerevisiae, in which inactivation of the two eIF5A genes (8), or of the single deoxyhypusine synthase gene (9, 10), causes a loss of cell viability. Deoxyhypusine synthase is a tetrameric enzyme consisting of four identical subunits (11). The crystal structure of the human enzyme in a complex with its cofactor, NAD, revealed four active sites at the interfaces of subunit dimers (11). The enzyme catalyzes a complex reaction normally involving two substrates, the polyamine spermidine (the donor of the butylamine moiety) and the protein substrate, the eIF5A precursor (the acceptor of the butylamine moiety), and a cofactor, NAD. We have established that deoxyhypusine synthesis occurs by way of a covalent enzyme-imine intermediate (12) in four steps (Scheme 1, left-hand pathway): (i) NAD-dependent dehydrogenation of s...

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Fluorinated analogues of spermidine as substrates of spermine synthase

Cited by 29 publications

References 20 publications

Inhibition of mammalian spermine synthase by N‐alkylated‐1,3‐diaminopropane derivatives in vitro and in cultured rat hepatoma cells

Inhibition of mammalian spermine synthase by N‐alkylated‐1,3‐diaminopropane derivatives in vitro and in cultured rat hepatoma cells

Determination of protonation constants of some fluorinated polyamines by means of 13 C NMR data processed by the new computer program HypNMR2000. Protonation sequence in polyamines

Reversal of the Deoxyhypusine Synthesis Reaction

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