[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.

[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.

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.

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.

[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.

[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.

[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 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.

Complexes of aspartate aminotransferase with hydroxylamine derivatives: spectral studies in solution and in the crystalline state.

Hydroxylamine and its derivatives of general formula H2NOR react with aldehydes and aldimines to produce oximes. If R corresponds to the side chain of a natural amino acid, such compounds can be thought of as analogs of the corresponding amino acids, lacking the alpha-carboxylate group. Oximes formed between such compounds and pyridoxal phosphate in the active site of aspartate amino-transferase mimic external aldimine intermediates that occur during catalysis by this enzyme. The properties of oxime derivatives of mitochondrial aspartate aminotransferase with hydroxylamine and 6 compounds H2NOR were studied by absorption spectroscopy and circular dichroism in solution and by linear dichroism in crystals. Stable oximes, absorbing at lambda max congruent to 380 nm and exhibiting a negative Cotton effect, were obtained with the carboxylate-containing compounds. The oximes formed with carboxylate-free compounds showed somewhat different properties and stability. With H-Tyr a stable complex absorbing at lambda max congruent to 370 nm rather than at 380 nm, was obtained, H-Ala and H-Phe produced unstable oximes with the initial absorption band at lambda max congruent to 380 nm that was gradually replaced by a band at lambda max congruent to 340 nm. The species absorbing at 340 nm were shown to be coenzyme-inhibitor complexes which were gradually released from the enzyme. A similar 330-340 nm absorption band was observed upon reaction of the free coenzyme with all hydroxylamine inhibitors at neutral pH-values. The results of the circular dichroism experiments in solution and the linear dichroism studies in microcrystals of mAspAT indicate that the coenzyme conformation in these inhibitor/enzyme complexes is similar to that occurring in an external aldimine analogue, the 2-MeAsp/mAspAT complex. Co-crystallizations of the enzyme with the H2NOR compounds were also carried out. Triclinic crystals were obtained in all cases, suggesting that the "closed" structure cannot be stabilized by a single carboxylate group.

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.

Aminooxy analogues of spermidine as inhibitors of spermine synthase and substrates of hepatic polyamine acetylating activity.

Aminooxy analogues of spermidine, 1-aminooxy-3-N-[3-aminopropyl]- aminopropane (AP-APA) and N-[2-aminooxyethyl]-1,4-diaminobutane (AOE-PU), were tested as substrates or inhibitors of the enzymes involved in methionine and polyamine metabolism. Both compounds were good competitive inhibitors and poor substrates of spermine synthase, good substrates of cytosolic polyamine acetyltransferase, inactivators of S-adenosylmethionine decarboxylase and inhibitors of ornithine decarboxylase. AP-APA and AOE-PU showed K1-values of 1.5 and 186 microM as inhibitors of purified spermine synthase, and Km-values of 1.4 and 2.1 mM as substrates of the crude hepatic polyamine acetyltransferase activity. AP-APA was more potent than AOE-PU in crude enzyme preparations. Neither drug had any significant effect at 1 mM concentration on the activities of spermidine synthase, methionine adenosyltransferase, S-adenosylhomocysteine hydrolase, and methylthioadenosine phosphorylase. The results suggest that compounds of this type are valuable tools in unraveling the physiology of polyamines.

Uptake of 3H-labeled 1-aminooxy-3-aminopropane by baby hamster kidney cells.

The uptake, catabolism, and release of H-labeled 1-aminooxy-3-aminopropane, a new putrescine analog shown to be a potent polyamine antimetabolite, into and from baby hamster kidney cells (BHK21/C13) were studied. The results show that [3H]-1-aminooxy-3-aminopropane (APA) is not concentrated in the cell, does not compete with polyamines for transport and reveals no difference in uptake between polyamine-depleted and control cells. After a 12-h culture, 60% of APA was recovered intact in the culture media. At this time point, only 30% of the intracellular radioactivity was intact APA, showing that the drug is catabolized in the cells. This intracellular ratio persisted throughout the 4-day culture period. The metabolites of APA were not characterized further. The results indicate that the drug is not recognized as a polyamine by the cells and does not replace or interfere with the polyamines in cellular functions. Thus, its potent affinity to ornithine decarboxylase and spermidine synthase is likely to be due to close structural similarity with the intermediates formed in these reactions. This has implications for the mechanisms involved.

Aminooxy-analogues of spermidine: new partial agonists and antagonists at the polyamine site of the rat hippocampal NMDA receptor complex.

The amino-1-oxy- and amino-8-oxy-analogues of spermidine (1-O-SPD and 8-O-SPD) were tested in vitro with rat hippocampal membranes as potential modulators of the N-methyl-D-aspartate (NMDA) receptor complex via the polyamine regulatory site. In the presence of 1 microM glutamate and glycine, the binding of the NMDA channel ligand [3H]MK-801 was stimulated by 8-O-SPD (EC50 = 50 microM); 1-O-SPD was without significant influence at concentrations up to 1 mM. Addition of 2 and 4 microM of the polyamine agonist spermine eliminated the stimulatory property of 8-O-SPD, whereas 1-O-SPD was inhibitory under these conditions (IC50 = 274 and 481 microM, respectively). At higher concentrations of spermine, both compounds were inhibitory. Inhibition of [3H]MK-801 binding by the inverse polyamine agonists 1,10-diaminodecane, 1,12-diaminododecane, and arcaine was attenuated by 1 mM 1-O-SPD. The data are compatible with the notion that 8-O-SPD is a partial polyamine agonist and that 1-O-SPD is an antagonist without intrinsic activity.

Aminooxy analogues of spermidine evidence the divergent roles of the charged amino nitrogens in the cellular physiology of spermidine.

Two recently devised spermidine analogues, N-[2-aminooxyethyl]-1,4-diaminobutane (AOEPU) and 1-aminooxy-3-N-[3-aminopropyl]-aminopropane (APAPA), were used to elucidate the role of charge distribution in the functions of spermidine in cultured baby hamster kidney cells. The drugs did not affect cell proliferation nor did they relieve the growth-arrest but potentiated the metabolic disturbances caused by DL-alpha-difluoromethyl-ornithine (DFMO). Neither drug affected spermidine uptake but both competed with putrescine uptake. Neither drug could replace spermidine in the control of S-adenosylmethionine decarboxylase and accumulation of the reaction product. APAPA prevented spermine synthesis and showed that modest putrescine synthesis take place in the presence of DFMO. AOEPU, but not APAPA, interfered with cellular constituents resulting in enzymatic formation, accumulation and excretion to culture medium of UV-absorbing catabolites.

The reduction of thymine residues in DNA by the combined action of UV light and hypophosphite.

UV irradiation (lambda = 254 nm) of thymine and uracil in aqueous solution containing salts of phosphinic acid (hypophosphites) results in the formation of the corresponding dihydropyrimidine derivatives. The peculiarities of this new photochemical reaction consist of a specificity towards 2,4-dioxophyrimidines, a high quantum yield and a neutral pH optimum. The quantum yield of photoconversion of thymine at pH 7.0 is equal to 1.9 x 10(-2) in 1 M NaH2PO2; it is diminished to 8.5 x 10(-3) in 0.1 M NaH2PO2. The same decrease in quantum yield with concentration is also found for uracil and uridine; quantum yields of their transformations in 0.1 M NaH2PO2 at pH 7.0 are 6.6 x 10(-2) and 1.5 x 10(-1) respectively. The mild conditions and high quantum yields characteristic of the photoinduced reaction above open up the possibility of obtaining DNA molecules which contain among pyrimidine photoproducts mainly dihydropyrimidine residues. A correlation between various types of thymine modifications in UV-irradiated double-stranded DNA (dimers and dihydrothymidine residues) and the amplitude of the intense negative band in the circular dichroism (CD) spectra specific for DNA liquid crystalline dispersions has been established. A possible application of this base-specific modification to the investigation of nucleic acids is considered.

[An effective inhibitor of S-adenosylmethionine decarboxylase]. / Effektivnyi ingibitor dekarboksilazy S-adenozilmetionina.

New analogues of cAMP, namely 2'-phosphate of cAMP and its esters, were obtained by direct phosphorylation of N6-benzoyladenosine 3',5'-cyclic phosphate with 2-cyanoethyl phosphate in the presence of DCC followed by the removal of protecting groups.