RESUMO
The biogenic polyamines, spermine (Spm) and spermidine, are organic polycations present in millimolar concentrations in all eukaryotic cells participating in the regulation of vital cellular functions including proliferation and differentiation. The design and biochemical evaluation of polyamine analogues are cornerstones of polyamine research. Here we synthesized and studied novel C-methylated Spm analogues: 2,11-dimethylspermine (2,11-Me2Spm), 3,10-dimethylspermine (3,10-Me2Spm), 2-methylspermine, and 2,2-dimethylspermine. The tested analogues overcame growth arrest induced by a 72 h treatment with α-difluoromethylornithine, an ornithine decarboxylase (ODC) inhibitor, and entered into DU145 cells via the polyamine transporter. 3,10-Me2Spm was a poor substrate of spermine oxidase and spermidine/spermine-N1-acetyltransferase (SSAT) when compared with 2,11-Me2Spm, thus resembling 1,12-dimethylspermine, which lacks the substrate properties required for the SSAT reaction. The antizyme (OAZ1)-mediated downregulation of ODC and inhibition of polyamine transport are crucial in the maintenance of polyamine homeostasis. Interestingly, 3,10-Me2Spm was found to be the first Spm analogue that did not induce OAZ1 and, consequently, was a weak downregulator of ODC activity in DU145 cells.
Assuntos
Inibidores da Ornitina Descarboxilase/farmacologia , Ornitina Descarboxilase/química , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Poliaminas/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Espermina/análogos & derivados , Espermina/metabolismo , Transporte Biológico , Metilação de DNA , Humanos , Masculino , Ornitina Descarboxilase/metabolismo , Neoplasias da Próstata/metabolismo , Especificidade por Substrato , Células Tumorais Cultivadas , Poliamina OxidaseRESUMO
Eukaryotic initiation factor 5A (eIF5A) has recently been identified as a biomarker of prognostic significance and therapeutic potential for the treatment in hepatocellular carcinoma. This prompted us to establish a rapid and robust assay to determine deoxyhypusine and hypusine formed with the purified enzymes deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH) from Plasmodium to develop a rapid screening assay for antimalarial drugs. The peptide hydrolysate obtained from hypusinylated eIF5A was analyzed by ultra performance liquid chromatography (UPLC) with retention times for deoxyhypusine of 7.44 min and for hypusine of 7.30 min, respectively. The limit of detection for both compounds was 0.144 ng/µl. Determination of the specific activity of Plasmodium DOHH resulted in a twofold higher specific activity than its human counterpart. Following the iron-complexing strategy of the ferrous iron which is present in the active site of Plasmodium DOHH, a series of iron chelating compounds was tested. 2,2'-Dipyridyl and mimosine abolished DOHH activity completely while 4-oxo-piperidine-carboxylates i.e. the nitrophenylether JK8-2 and EHW 437, the oxime ether of the piperidine aldehyde, showed no inhibition although they were highly active in in vitro cultures of Plasmodium and in vivo in a rodent mouse model. The method allows a high-throughput screening (HPTS) of antimalarial drugs and the evaluation of eIF5A as a biomarker.
Assuntos
Antimaláricos/isolamento & purificação , Fator de Iniciação 5 em Eucariotos/metabolismo , Lisina/análogos & derivados , Malária/tratamento farmacológico , Plasmodium/enzimologia , Animais , Antimaláricos/química , Antimaláricos/uso terapêutico , Domínio Catalítico , Cromatografia Líquida de Alta Pressão , Fator de Iniciação 5 em Eucariotos/genética , Fator de Iniciação 5 em Eucariotos/isolamento & purificação , Humanos , Lisina/isolamento & purificação , Lisina/metabolismo , Malária/diagnóstico , Camundongos , Oxigenases de Função Mista/isolamento & purificação , Oxigenases de Função Mista/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/isolamento & purificação , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Hidrolisados de Proteína/químicaRESUMO
The polyamines, putrescine, spermidine, and spermine, are ubiquitous multifunctional cations essential for cellular proliferation. One specific function of spermidine in cell growth is its role as a butylamine donor for hypusine synthesis in the eukaryotic initiation factor 5A (eIF5A). Here, we report the ability of novel mono-methylated spermidine analogs (α-MeSpd, ß-MeSpd, γ-MeSpd, and ω-MeSpd) to function in the hypusination of eIF5A and in supporting the growth of DFMO-treated DU145 cells. We also tested them as substrates and inhibitors for deoxyhypusine synthase (DHS) in vitro. Of these compounds, α-MeSpd, ß-MeSpd, and γ-MeSpd (but not ω-MeSpd) were substrates for DHS in vitro, while they all inhibited the enzyme reaction. As racemic mixtures, only α-MeSpd and ß-MeSpd supported long-term growth (9-18 days) of spermidine-depleted DU145 cells, whereas γ-MeSpd and ω-MeSpd did not. The S-enantiomer of α-MeSpd, which supported long-term growth, was a good substrate for DHS in vitro, whereas the R-isomer was not. The long-term growth of DFMO-treated cells correlated with the hypusine modification of eIF5A by intracellular methylated spermidine analogs. These results underscore the critical requirement for hypusine modification in mammalian cell proliferation and provide new insights into the specificity of the deoxyhypusine synthase reaction.
Assuntos
Divisão Celular/efeitos dos fármacos , Lisina/análogos & derivados , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Espermidina/farmacologia , Western Blotting , Linhagem Celular Tumoral , Humanos , Lisina/metabolismo , Masculino , Metilação , Espermidina/análogos & derivados , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
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.
Assuntos
Poliaminas/metabolismo , Espermidina/análogos & derivados , Espermidina/síntese química , Acetiltransferases/química , Acetiltransferases/genética , Processamento Alternativo , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Eflornitina/farmacologia , Humanos , Camundongos , Inibidores da Ornitina Descarboxilase , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/química , Precursores de RNA/genética , RNA Mensageiro/genética , Proteínas Recombinantes/química , Espermidina/química , Espermidina/farmacologia , Relação Estrutura-AtividadeRESUMO
Earlier unknown racemic ß-methylspermidine (ß-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 ß- and γ-MeSpd's. 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 ß- and γ-MeSpd's in combination with earlier α-MeSpd are useful tools for studying enzymology and cell biology of polyamines.
Assuntos
Bioquímica/métodos , Espermidina/metabolismo , 1-Butanol/síntese química , Metilação , Nitrilas/síntese química , Espermidina/químicaRESUMO
The earliest studies concerning polyamines (PAs) in plants were performed by using in vitro cultured explants of Helianthus tuberosus dormant tuber. This parenchyma tissue was particularly useful due to its susceptibility to several growth substances, including PAs. During tuber dormancy, PA levels are too low to sustain cell division; thus Helianthus represents a natural PA-deficient model. When cultivated in vitro in the presence of auxins, Helianthus tuber dormant parenchyma cells at the G(0) stage start to divide synchronously acquiring meristematic characteristics. The requirement for auxins to induce cell division can be substituted by aliphatic PAs such as putrescine, spermidine or spermine. Cylinders or slices of explanted homogeneous tuber parenchyma were cultured in liquid medium for short-term studies on the cell cycle, or on solid agar medium for long-term experiments. Morphological and physiological modifications of synchronously dividing cells were studied during the different phases of the cell cycle in relation to PAs biosynthesis and oxidation. Long-term experiments led to the identification of the PAs as plant growth regulators, as the sole nitrogen source, as tuber storage substances and as essential factors for morphogenetic processes and cell homeostasis. More recently this system was used to study the effects on plant cell proliferation of platinum- or palladium-derived drugs (cisplatin and platinum or palladium bi-substituted spermine) that are used in human cancer cell lines as antiproliferative and cytotoxic agents. Cisplatin was the most active both in cell proliferation inhibition and on PA metabolism. Similar experiments were performed using three agmatine analogous. Different effects of these compounds were observed on cell proliferation, free PA levels and enzyme activities, leading to a hypothesis of a correlation between their chemical structure and the agmatine metabolism in plants.
Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Helianthus/fisiologia , Compostos de Platina/farmacologia , Poliaminas/metabolismo , Agmatina/metabolismo , Agmatina/farmacologia , Animais , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Helianthus/efeitos dos fármacos , Helianthus/crescimento & desenvolvimento , Humanos , Neoplasias , Paládio , Reguladores de Crescimento de Plantas/metabolismo , Reguladores de Crescimento de Plantas/farmacologia , Poliaminas/farmacologiaRESUMO
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, alpha-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.
Assuntos
Ésteres/química , Ésteres/farmacologia , Hidroxilamina/síntese química , Hidroxilamina/farmacologia , Antiprotozoários/síntese química , Antiprotozoários/química , Antiprotozoários/farmacologia , Ésteres/síntese química , Humanos , Hidroxilamina/química , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/crescimento & desenvolvimento , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/parasitologia , Propilaminas/farmacologiaRESUMO
The efficacy of gamma-guanidinooxypropylamine (GAPA), a novel agmatine analogue against protozoan parasite, Leishmaniadonovani was evaluated. Wild-type and ornithine decarboxylase-overexpressors of L. donovani were used to study the effect and mode of action of this inhibitor. GAPA inhibited the growth of both promastigotes and amastigotes. Ornithine decarboxylase (ODC) activity and polyamine levels were markedly lower in cells treated with GAPA and proliferation was rescued by addition of putrescine or spermidine. GAPA inhibited L. donovani recombinant ODC with K(i) value of approximately 60microM. The ODC-overexpressors showed significant resistance to GAPA. GAPA has pK(a) 6.71 and at physiological pH the analogue can mimic protonated state of putrescine and can probably use putrescine transport system. Transport of putrescine in wild-type L. donovani promastigotes was inhibited by GAPA. We for the first time report that GAPA is a potential antileishmanial lead compound and it possibly inhibits L. donovani growth by depletion of intracellular polyamine levels.
Assuntos
Agmatina/farmacologia , Antiprotozoários/farmacologia , Poliaminas Biogênicas/antagonistas & inibidores , Proliferação de Células/efeitos dos fármacos , Guanidinas/farmacologia , Leishmania donovani/efeitos dos fármacos , Propilaminas/farmacologia , Animais , Poliaminas Biogênicas/metabolismo , Transporte Biológico/efeitos dos fármacos , Guanidinas/química , Leishmania donovani/citologia , Leishmania donovani/metabolismo , Propilaminas/químicaRESUMO
New mimics of the transition state of spermine/spermidine-N(1)-acetyltransferase reaction were prepared starting from aminooxy analogues of spermidine or spermine and SH-CoA. The activity depended on the structure of polyamine fragment of the conjugate and best of the synthesized compounds were active at micromolar concentrations.