[Interaction of DNA Methyltransferase Dnmt3a with Phosphorus Analogs of S-Adenosylmethionine and S-Adenosylhomocysteine].

Enzymatic methyltransferase reactions are of crucial importance for cell metabolism. S-Adenosyl-L-methionine (AdoMet) is a main donor of the methyl group. DNA, RNA, proteins, and low-molecular-weight compounds are substrates of methyltransferases. In mammals, DNA methyltransferase Dnmt3a de novo methylates the C5 position of cytosine residues in CpG sequences in DNA. The methylation pattern is one of the factors that determine the epigenetic regulation of gene expression. Here, interactions with the catalytic domain of Dnmt3a was for the first time studied for phosphonous and phosphonic analogs of AdoMet and S-adenosyl-L-homocysteine (AdoHcy), in which the carboxyl group was substituted for respective phosphorus-containing group. These AdoMet analogs were shown to be substrates of Dnmt3a, and the methylation efficiency was only halved as compared with that of natural AdoMet. Both phosphorus-containing analogs of AdoHcy, which is a natural methyltransferase inhibitor, showed similar inhibitory activities toward Dnmt3a and were approximately four times less active than AdoHcy. The finding that the phosphonous and phosphonic analogs are similar in activity was quite unexpected because the geometry and charge of their phosphorus-containing groups differ substantially. The phosphorus-containing analogs of AdoMet and AdoHcy are discussed as promising tools for investigation of methyltransferases.

Identification of a Novel Substrate-Derived Spermine Oxidase Inhibitor.

Homeostasis of the biogenic polyamines spermine (Spm) and spermidine (Spd), present in µM-mM concentrations in all eukaryotic cells, is precisely regulated by coordinated activities of the enzymes of polyamine synthesis, degradation, and transport, in order to sustain normal cell growth and viability. Spermine oxidase (SMOX) is the key and most recently discovered enzyme of polyamine metabolism that plays an essential role in regulating polyamine homeostasis by catalyzing the back-conversion of Spm to Spd. The development of many types of epithelial cancer is associated with inflammation, and disease-related inflammatory stimuli induce SMOX. MDL72527 is widely used in vitro and in vivo as an irreversible inhibitor of SMOX, but it is also potent towards N1-acetylpolyamine oxidase. Although SMOX has high substrate specificity, Spm analogues have not been systematically studied as enzyme inhibitors. Here we demonstrate that 1,12-diamino-2,11-bis(methylidene)-4,9-diazadodecane (2,11-Met2-Spm) has, under standard assay conditions, an IC50 value of 169 µM towards SMOX and is an interesting instrument and lead compound for studying polyamine catabolism.

[Enantioselective Synthesis of (R)- and (S)-3-Methylspermidines].

Earlier unknown enantiomerically pure (R)- and (S)-1,8-diamino-3-methyl-4-azaoctane's (3-MeSpd's) were synthesized with high overall yields and optical purity starting from commercially available R- and S-isomers of N-Boc-2-aminopropanol-1. Application of R- and S-isomers of 3-MeSpd for the investigation of the stereospecificity of spermidine transporter and peculiarities of deoxyhypusine synthase reaction are discussed.

Hydroxylamine derivatives for regulation of spermine and spermidine metabolism.

The biogenic polyamines spermine, spermidine, and their precursor putrescine are present in micro-to-millimolar concentrations in all cell types and are vitally important for their normal growth. High intracellular content of spermine and spermidine determines the multiplicity of the cellular functions of the polyamines. Many of these functions are not well characterized at the molecular level, ensuring the ongoing development of this field of biochemistry. Tumor cells have elevated polyamine level if compared with normal cells, and this greatly stimulates the search for new opportunities to deplete the intracellular pool of spermine and spermidine resulting in decrease in cell growth and even cell death. O-Substituted hydroxylamines occupy their own place among chemical regulators of the activity of the enzymes of polyamine metabolism. Varying the structure of the alkyl substituent made it possible to obtain within one class of chemical compounds highly effective inhibitors and regulators of the activity of all the enzymes of putrescine, spermine and spermidine metabolism (with the exception of FAD-dependent spermine oxidase and acetylpolyamine oxidase), effectors of the polyamine transport system, and even actively transported in cells "proinhibitor" of ornithine decarboxylase. Some principles for the design of specific inhibitors of these enzymes as well as the peculiarities of cellular effects of corresponding O-substituted hydroxylamines are discussed.

Biogenic polyamines spermine and spermidine activate RNA polymerase and inhibit RNA helicase of hepatitis C virus.

Influence of the biogenic polyamines spermine, spermidine, and putrescine as well as their derivatives on the replication enzymes of hepatitis C virus (HCV) was investigated. It was found that spermine and spermidine activate HCV RNA-dependent RNA polymerase (NS5B protein). This effect was not caused by the stabilization of the enzyme or by competition with template-primer complex, but rather it was due to achievement of true maximum velocity V(max). Natural polyamines and their derivatives effectively inhibited the helicase reaction catalyzed by another enzyme of HCV replication - helicase/NTPase (NS3 protein). However, these compounds affected neither the NTPase reaction nor its activation by polynucleotides. Activation of the HCV RNA polymerase and inhibition of the viral helicase were shown at physiological concentrations of the polyamines. These data suggest that biogenic polyamines may cause differently directed effects on the replication of the HCV genome in an infected cell.

Inhibition of DNA gyrase by levofloxacin and related fluorine-containing heterocyclic compounds.

Fluoroquinolones are an important class of modern and efficient antibacterial drugs with a broad spectrum of activity. Levofloxacin (the optically active form of ofloxacin) is one of the most promising fluoroquinolone drugs, and its antibacterial activity is substantially higher than the activity of other drugs of the fluoroquinolone family. Earlier, in the Postovsky Institute of Organic Synthesis, UB RAS, an original method of levofloxacin synthesis was developed, and now the pilot batch of the drug is being prepared. Bacterial DNA gyrase is a specific target of fluoroquinolones; hence, the study of the enzyme-drug interaction is of theoretical and practical importance. Moreover, the parameters of DNA gyrase inhibition may serve as a criterion for drug quality. Here, we present the results of studying the interaction of DNA gyrase with a number of fluoroquinolones and their analogs: intermediates and semi-products of the levofloxacin synthesis, and also samples from the pilot batches of this drug. The importance of two structural elements of the levofloxacin molecule for the efficiency of the inhibition is revealed. The data obtained may be useful for the design of new drugs derived from levofloxacin.

Acute pancreatitis induced by activated polyamine catabolism is associated with coagulopathy: effects of alpha-methylated polyamine analogs on hemostasis.

BACKGROUND/AIMS: Polyamines are ubiquitous organic cations essential for cellular proliferation and tissue integrity. We have previously shown that pancreatic polyamine depletion in rats overexpressing the catabolic enzyme, spermidine/spermine N(1)-acetyltransferase (SSAT), results in the development of severe acute pancreatitis, and that therapeutic administration of metabolically stable alpha-methylated polyamine analogs protects the animals from pancreatitis-associated mortality. Our aim was to elucidate the therapeutic mechanism(s) of alpha-methylspermidine (MeSpd). METHODS: The effect of MeSpd on hemostasis and the extent of organ failure were studied in SSAT transgenic rats with either induced pancreatitis or lipopolysaccharide (LPS)-induced coagulopathy. The effect of polyamines on fibrinolysis and coagulation was also studied in vitro. RESULTS: Pancreatitis caused a rapid development of intravascular coagulopathy, as assessed by prolonged coagulation times, decreased plasma fibrinogen level and antithrombin activity, enhanced fibrinolysis, reduced platelet count and presence of schistocytes. Therapeutic administration of MeSpd restored these parameters to almost control levels within 24 h. In vitro, polyamines dose-dependently inhibited fibrinolysis and intrinsic coagulation pathway. In LPS-induced coagulopathy, SSAT transgenic rats were more sensitive to the drug than their syngeneic littermates, and MeSpd-ameliorated LPS-induced coagulation disorders. CONCLUSION: Pancreatitis-associated mortality in SSAT rats is due to coagulopathy that is alleviated by treatment with MeSpd.

[Methylated analogues of spermine and spermidine as tools to investigate cellular functions of polyamines and the enzymes of their metabolism].

Biogenic amines spermine and spermidine are essential factors of cellular growth. Polyamine analogues are widely used to investigate and to regulate the enzymes of polyamine metabolism and functions of spermine and spermidine in vitro and in vivo. Recently, it was demonstrated that alpha-methylated derivatives of spermine and spermidine are capable to fulfill key cellular functions of polyamines, moreover in some cases of (R)- and (S)-isomers are actually different. Using these alpha-methylated spermine and spermidine analogues it turned possible to prevent the development of acute pancreatitis of SSAT-transgenic rats and to demostrate for the first time that polyamine oxidase, spermine oxidase and deoxyhypusine synthase have dormant stereospecificity. An original approach to regulate the stereospecificity of polyamine oxidase was suggested. It was also demonstrated that the depletion of the intracellular polyamine pool has both hypusine-related consequences and also the consequences unrelated to the posttranslational modification of eukaryotic initiation translation factor eIF5A. Possible applications of a new family of C-methylated polyamine analogues for the investigation and regulation of polyamine metabolism in vitro and in vivo are discussed.

Novel approach to design an isosteric charge-deficient analogue of spermine and its biochemically important derivatives.

The design and synthesis of SpmTrien (1,12-diamino-3,6,9-triazadodecane), an isosteric and charge-deficient analogue of spermine with excellent chelating properties towards Cu(2+) ions, as well as novel N(1)- and N(12)-Ac-SpmTriens and bis-Et-SpmTrien (N(1),N(12)-diethyl-1,12-diamino-3,6,9-triazadodecane) are described. Possible applications of SpmTrien and its derivatives to the investigation of the enzymes of polyamine metabolism and spermine cellular functions, including interaction with DNA, are discussed.

Inhibition of agmatine transport in liver mitochondria by new charge-deficient agmatine analogues.

The charge of the agmatine analogues AO-Agm [N-(3-aminooxypropyl)guanidine], GAPA [N-(3-aminopropoxy)guanidine] and NGPG [N-(3-guanidinopropoxy)guanidine] is deficient as compared with that of agmatine and they are thus able to inhibit agmatine transport in liver mitochondria. The presence of the guanidine group is essential for an optimal effect, since AO-Agm and NGPG display competitive inhibition, whereas that of GAPA is non-competitive. NGPG is the most effective inhibitor (K(i)=0.86 mM). The sequence in the inhibitory efficacy is not directly dependent on the degree of protonation of the molecules; in fact NGPG has almost the same charge as GAPA. When the importance of the guanidine group for agmatine uptake is taken into account, this observation suggests that the agmatine transporter is a single-binding, centre-gated pore rather than a channel.

[Aminooxy analogues of spermine and their monoacetyl derivatives].

Convenient methods of synthesis of 1-aminooxy-3,8-diaza-11-aminoundecane, its earlier unknown N1-and N1 -acetyl derivatives, and also 1,10-bis(aminooxy)-3,8-diazadecane are suggested. It is shown a possibility to selectively delete the acid-labile ethoxyethylidene protection of aminooxy group by hydrosulfates in the presence of N-tert-butyloxycarbonyl group.

[New charge-deficient agmatine analogs].

N,N'-Di-Boc-N"-triflylguanidine was demonstrated to be an efficient guanidinylation reagent for O-substituted hydroxylamines. N-(3-Aminooxypropyl)- and N-(3-aminopropoxy)guanidines, previously unknown isosteric and charge-deficient agmatine analogues, have been synthesized. The possibilities of using these compounds in studying polyamine metabolism are discussed. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 6; see also http://www.maik.ru.

[A charge-deficient analogue of spermine with chelating properties].

1,12-Diamino-3,6,9-triazadodecane, a new isosteric and charge-deficient analogue of spermine, is synthesized. Unlike spermine, the new analogue is an excellent chelator of Cu2+ ions. Possible applications of this compound for studying enzymes of polyamine metabolism and cellular functions of spermine are discussed. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 3; see also http://www.maik.ru.

[New oxaanalogues of spermine].

A new isosteric charge-deficient spermine analogue, 1,12-diamino-4,9-diaza-5-oxadodecan, and O-(7-amino-4-azaheptyl)oxime of 3-aminopropanal, a stable analogue of the Schiff base intermediate in the enzymatic oxidation of spermine, were synthesized. The possible use of these compounds for the inhibition of spermine oxidase is discussed.

[New syntheses of alpha-methyl- and alpha,alpha'-dimethylspermine].

alpha-Methylspermine and alpha,alpha'-dimethylspermine were synthesized in high overall yields starting from N-(benzyloxycarbonyl)-3-aminobutanol in order to study polyamine biochemistry in vitro and in vivo.

[A new synthesis of alpha-methylspermidine]. / Novyi sintez alpha-metilspermidina.

A five-step synthesis of alpha-methylspermidine (1,8-diamino-5-azanonane), the first polyamine analogue preventing pathological consequences of spermidine depletion in transgenic rats overproducing spermine/spermidine N'-acetyltransferase, from ethyl 3-aminobutyrate was achieved in a high overall yield.

Inhibition of enzymes of polyamine biosynthesis by substrate-like O-substituted hydroxylamines.

The biogenic amines spermine, spermidine, and putrescine are essential factors of cell growth and differentiation. To inhibit pyridoxal-5'-phosphate dependent ornithine decarboxylase and pyruvate dependent S-adenosylmethionine decarboxylase, key enzymes of polyamine biosynthesis, a system of substrate-like O-substituted hydroxylamines is suggested. The best of these compounds were active at nanomolar concentrations. High potency and specificity of this type of inhibitors are discussed in terms of structural similarity of E-I and E-S complexes.

Hydroxylamine-containing inhibitors of polyamine biosynthesis and impairment of colon cancer cell growth.

Polyamine synthesis (by the action of ornithine decarboxylase [ODC] and S-adenosylmethionine decarboxylase [SAMDC]) and polyamine content are high in colon cancer. In addition, colonic lumen is rich in polyamines synthesised by colonic microflora; for this reason, polyamine depletion in colon cancer may be a logical approach to impair growth of colon cancer cells. We evaluated highly specific and reportedly non-toxic hydroxylamine-containing inhibitors of ODC (1-aminooxy-3-aminopropane, APA) and SAMDC (S-(5'-deoxy-5'-adenosyl)-methylthioethyl-hydroxylamine, AMA) in human colon cancer cells (Caco-2 and HT-29) in culture. APA depleted ODC activity within 24 hr, more rapidly than did difluoromethylornithine. APA and AMA in combination (100 microM each) reduced ODC and SAMDC activities to undetectable levels within 24 hr and intracellular polyamines to 8-23% of control. The resulting growth arrest could be reversed only by twice as much spermidine as is physiologically present in the colonic lumen. In concentrations sufficient to deplete growth, APA and AMA were not toxic. Simultaneous treatment with APA, AMA, and 5-fluorouracil reduced colon cancer cell survival more potently than treatment with 5-fluorouracil alone. The hydroxylamine-containing ODC and SAMDC inhibitors APA and AMA are potent inhibitors of colon cancer cell proliferation and might be therapeutically promising in colon cancer.

[Phosphinic analog of methionine inhibits growth of leucosis cell L1210 and transforms to phosphinic analog of S-adenosylmethionine]. / Fosfinovyi analog metionina tormozit rost leikoznykh kletok L1210 i prevrashchaetsia v fosfinovyi analog S-adenozilmetionina.

A phosphinic analogue of methionine bearing a phosphinic H(OH)(O)P fragment in place of the carboxyl group inhibited the growth of the L1210 cells and was intracellularly transformed to the phosphinic analogue of S-adenosylmethionine.

[Acyclic nucleotide analogues based on phosphonic acids]. / Atsiklicheskie analogi nukleotidov na osnove fosfonovykh kislot.

The synthesis of novel nucleotide analogues on the basis of ethylphosphonic acid was described. A rigid structural element, an amide or a double bond, was characteristics of the compounds synthesized. The antiviral and cytotoxic activities of these compounds were studied in cell cultures.