M. A. Khomutov

The Use of Novel C-Methylated Spermidine Derivatives To Investigate the Regulation of Polyamine Metabolism

The polyamines are organic polycations present at millimolar concentrations in eukaryotic cells where they participate in the regulation of vital cellular functions including proliferation and differentiation. Biological evaluation of rationally designed polyamine analogs is one of the cornerstones of polyamine research. Here we have synthesized and characterized novel C-methylated spermidine analogs, that is, 2-methylspermidine, 3-methylspermidine, and 8-methylspermidine. 3-Methylspermidine was found to be metabolically stable in DU145 cells, while 8-methylspermidine was a substrate for spermidine/spermine N(1)-acetyltransferase (SSAT) and 2-methylspermidine was a substrate for both SSAT and acetylpolyamine oxidase. All the analogs induced the splicing of the productive mRNA splice variant of SSAT, overcame growth arrest induced by 72-h treatment with ornithine decarboxylase (ODC) inhibitor α-difluoromethylornithine, and were transported via the polyamine transporter. Surprisingly, 2-methylspermidine was a weak downregulator of ODC activity in DU145 cells. Our data demonstrates that it is possible to radically alter the biochemical properties of a polyamine analog by changing the position of the methyl group.

Novel convenient synthesis of biologically active esters of hydroxylamine

Alkylation of ethyl N-hydroxyacetimidate with readily available methanesulfonates of functionally substituted alcohols and subsequent deprotection of aminooxy group is a novel and convenient method to prepare functionally substituted esters of hydroxylamine with high overall yield. This approach is a good alternative to well-known reaction of N-hydroxyphthalimide with alcohols under the Mitsunobu conditions. The properties of ethoxyethylidene protection of aminooxy group on the contrary to that of N-alkoxyphthalimide group allow to perform a wide spectra of the transformations in the radical of N-protected hydroxylamine derivatives. This is essential for synthetic strategies consisting in the introduction of N-protected aminooxy group at one of the first steps of synthesis and subsequent transformations of the radical.The inhibitory effect of one of the newly synthesized compound, 1-guanidinooxy-3-aminopropane (GAPA), was compared with that of well-known inhibitors of ornithine decarboxylase namely, α-difluoromethylornithine (DFMO) and 1-aminooxy-3-aminopropane (APA) on Leishmania donovani, a protozoan parasite that causes visceral leishmaniasis. GAPA, on the contrary with APA and DFMO, in micromolar concentrations, inhibited the growth of both amastigotes and promastigotes of sodium antimony gluconate-resistant forms of L. donovani.

Methylated analogs of spermine and spermidine as tools to investigate cellular functions of polyamines and enzymes of their metabolism

Biogenic amines spermine (Spm) and spermidine (Spd) are essential for cell growth. Polyamine analogs are widely used to investigate the enzymes of polyamine metabolism and the functions of spermine and spermidine in vitro and in vivo. It was demonstrated recently that α-methylated derivatives of Spm and Spd are able to fulfill the key cellular functions of polyamines, moreover, in some cases, the effects of (R) and (S) isomers were actually different. Using these α-methylated analogs of Spm and Spd, it turned possible to prevent the development of acute pancreatitis in SSAT-transgenic rats with controllable expression of the Spm/Spd N1-acetyltransferase gene. The analogs made it possible to reveal dormant stereospecificity of polyamine oxidase, Spm oxidase, and deoxyhypusine synthase. An original approach was suggested to regulate the stereospecificity of polyamine oxidase. Depletion of the intracellular polyamine pool was found to have both hypusine-related consequences and consequences unrelated to posttranslational modification of the eukaryotic translation initiation factor eIF5A. Possible applications of a new family of C-methylated polyamine analogs for the investigation and regulation of polyamine metabolism in vitro and in vivo 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 Vmax. 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.

Enantiomers of 3-methylspermidine selectively modulate deoxyhypusine synthesis and reveal important determinants for spermidine transport.

Eukaryotic translation initiation factor 5A (eIF5A) is essential for cell proliferation, becoming functionally active only after post-translational conversion of a specific Lys to hypusine [N(ε)-(4-amino-2-hydroxybutyl)lysine]. Deoxyhypusine synthase (DHS) is the rate-limiting enzyme of this two-step process, and the polyamine spermidine is the only natural donor of the butylamine group for this reaction, which is very conserved-hypusine biosynthesis suffers last when the intracellular spermidine pool is depleted. DHS has a very strict substrate specificity, and only a few spermidine analogs are substrates of the enzyme and can support long-term growth of spermidine-depleted cells. Herein, we compared the biological properties of earlier unknown enantiomers of 3-methylspermidine (3-MeSpd) in deoxyhypusine synthesis, in supporting cell growth and in polyamine transport. Long-term treatment of DU145 cells with α-difluoromethylornithine (inhibitor of polyamine biosynthesis) and (R)-3-MeSpd did not cause depletion of hypusinated eIF5A, and the cells were still able to grow, whereas the combination of α-difluoromethylornithine with a racemate or (S)-3-MeSpd caused cessation of cell growth. Noticeably, DHS preferred the (R)- over the (S)-enantiomer as a substrate. (R)-3-MeSpd competed with [(14)C]-labeled spermidine for cellular uptake less efficiently than the (S)-3-MeSpd (Ki = 141 μM vs 19 μM, respectively). The cells treated with racemic 3-MeSpd accumulated intracellularly mainly (S)-3-MeSpd, but not DHS substrate (R)-3-MeSpd, explaining the inability of the racemate to support long-term growth. The distinct properties of 3-MeSpd enantiomers can be exploited in designing polyamine uptake inhibitors, facilitating drug delivery and modulating deoxyhypusine synthesis.

Methylated Polyamines as Research Tools

Earlier unknown racemic β-methylspermidine (β-MeSpd) and γ-methylspermidine (γ-MeSpd) were -synthesized starting from crotononitrile or methacrylonitrile and putrescine. Lithium aluminum hydride reduction of the intermediate di-Boc-nitriles resulted in corresponding di-Boc-amines, which after deprotection gave target β- and γ-MeSpds. To prepare α-MeSpd, the starting compound, 3-amino-1-butanol, was converted into N-Cbz-3-amino-1-butyl methanesulfonate, which alkylated putrescine to give (after deprotection of amino group) the required α-MeSpd. Novel β- and γ-MeSpds in combination with earlier α-MeSpd are useful tools for studying enzymology and cell biology of polyamines.

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.

Novel metabolically stable and functionally active mimetic of spermidine

Earlier unknown 1,8-diamino-3-methyl-4-azanonane (γ-MeSpd) was synthesized. The analogue was a substrate of neither spermine/spermidine N1-acetyltransferase nor spermine synthase, but was capable to support the growth of DU145 cells having depleted polyamine pools. Such a combination of γ-MeSpd properties discloses novel opportunities to study cellular functions of catabolically unstable and easily interconvertible spermine and spermidine.

Leishmania donovani: structural insignt in the recognition of C-methylated analogues of spermidine as natural polyamines

The ability of α-, ν-, γ- and ω-methylated spermidine analogues to restore the growth of L. donovani promastigotes that were depleted of putrescine and spermidine was investigated. Only β-methylated spermidine, like natural spermidine, was capable of restoring the growth of L. donovani, while the remaining three analogues turned out to be inactive. α-Methylated spermidine is a functionally active spermidine surrogate both in vivo and in vitro, hence this analogue may be considered as an antidote in the host-parasite system, especially in the cases where inhibitors of polyamine biosynthesis are used for the therapy of leishmaniasis.

Aminooxy adsorbents derived from sephareose and toyopearl

Convenient synthetic schemes being suitable for the preparation of aminooxy-Sepharose and hydroxylamine-containing Toyopearl with capacity of 0.35–0.015 mmols of H2NO-groups per mL gel, including the adsorbent with easily cleavable disulfide bond in the linker are developed.