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1.
Molecules ; 28(3)2023 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-36770901

RESUMO

The fast spread of bacteria that are resistant to many classes of antibiotics (multidrug resistant) is a global threat to human and animal health with a worrisome scenario ahead. Novel therapeutical strategies are of crucial importance to combat this phenomenon. For this purpose, we investigated the antimicrobial properties of the naturally occurring tripeptide Bialaphos and a dipeptide L-leucyl-L-phosphinoithricin, the synthesis and diastereomers separation of which are herein described. We demonstrate that these compounds are effective on clinical isolates of the human pathogen Klebsiella pneumoniae, causing hospital-acquired and community-acquired infections. The tested isolates were remarkable for their resistance to more than 20 commercial antibiotics of different classes. Based on previous literature data and our experiments consisting of glutamine supplementation, we suggest that both compounds release phosphinothricin-a well-known nanomolar inhibitor of glutamine synthetase-after their penetration in the bacterial cells; and, in this way, exert their antibacterial effect by negatively affecting nitrogen assimilation in this pathogen.


Assuntos
Anti-Infecciosos , Infecções por Klebsiella , Humanos , Antibacterianos/farmacologia , Anti-Infecciosos/farmacologia , Farmacorresistência Bacteriana Múltipla , Infecções por Klebsiella/tratamento farmacológico , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae , Testes de Sensibilidade Microbiana , Peptídeos/farmacologia
2.
Int J Mol Sci ; 23(9)2022 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-35563006

RESUMO

The polyamines, spermine (Spm) and spermidine (Spd), are important for cell growth and function. Their homeostasis is strictly controlled, and a key downregulator of the polyamine pool is the polyamine-inducible protein, antizyme 1 (OAZ1). OAZ1 inhibits polyamine uptake and targets ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine biosynthesis, for proteasomal degradation. Here we report, for the first time, that polyamines induce dimerization of mouse recombinant full-length OAZ1, forming an (OAZ1)2-Polyamine complex. Dimerization could be modulated by functionally active C-methylated spermidine mimetics (MeSpds) by changing the position of the methyl group along the Spd backbone-2-MeSpd was a poor inducer as opposed to 1-MeSpd, 3-MeSpd, and Spd, which were good inducers. Importantly, the ability of compounds to inhibit polyamine uptake correlated with the efficiency of the (OAZ1)2-Polyamine complex formation. Thus, the (OAZ1)2-Polyamine complex may be needed to inhibit polyamine uptake. The efficiency of polyamine-induced ribosomal +1 frameshifting of OAZ1 mRNA could also be differentially modulated by MeSpds-2-MeSpd was a poor inducer of OAZ1 biosynthesis and hence a poor downregulator of ODC activity unlike the other MeSpds. These findings offer new insight into the OAZ1-mediated regulation of polyamine homeostasis and provide the chemical tools to study it.


Assuntos
Poliaminas , Espermidina , Animais , Dimerização , Mudança da Fase de Leitura do Gene Ribossômico , Camundongos , Ornitina Descarboxilase/metabolismo , Poliaminas/química , Poliaminas/metabolismo , Poliaminas/farmacologia , Proteínas , Espermidina/química , Espermidina/metabolismo , Espermidina/farmacologia
3.
J Biol Chem ; 295(10): 3247-3256, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-31996374

RESUMO

Snyder-Robinson syndrome (SRS) is an X-linked intellectual disability syndrome caused by a loss-of-function mutation in the spermine synthase (SMS) gene. Primarily affecting males, the main manifestations of SRS include osteoporosis, hypotonic stature, seizures, cognitive impairment, and developmental delay. Because there is no cure for SRS, treatment plans focus on alleviating symptoms rather than targeting the underlying causes. Biochemically, the cells of individuals with SRS accumulate excess spermidine, whereas spermine levels are reduced. We recently demonstrated that SRS patient-derived lymphoblastoid cells are capable of transporting exogenous spermine and its analogs into the cell and, in response, decreasing excess spermidine pools to normal levels. However, dietary supplementation of spermine does not appear to benefit SRS patients or mouse models. Here, we investigated the potential use of a metabolically stable spermine mimetic, (R,R)-1,12-dimethylspermine (Me2SPM), to reduce the intracellular spermidine pools of SRS patient-derived cells. Me2SPM can functionally substitute for the native polyamines in supporting cell growth while stimulating polyamine homeostatic control mechanisms. We found that both lymphoblasts and fibroblasts from SRS patients can accumulate Me2SPM, resulting in significantly decreased spermidine levels with no adverse effects on growth. Me2SPM administration to mice revealed that Me2SPM significantly decreases spermidine levels in multiple tissues. Importantly, Me2SPM was detectable in brain tissue, the organ most affected in SRS, and was associated with changes in polyamine metabolic enzymes. These findings indicate that the (R,R)-diastereomer of 1,12-Me2SPM represents a promising lead compound in developing a treatment aimed at targeting the molecular mechanisms underlying SRS pathology.


Assuntos
Deficiência Intelectual Ligada ao Cromossomo X/patologia , Espermidina/metabolismo , Espermina/análogos & derivados , Acetiltransferases/genética , Acetiltransferases/metabolismo , Animais , Encéfalo/metabolismo , Cromatografia Líquida de Alta Pressão , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Masculino , Deficiência Intelectual Ligada ao Cromossomo X/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Poliaminas/análise , Poliaminas/metabolismo , Espermina/administração & dosagem , Espermina/metabolismo , Espermina/farmacologia , Espermina Sintase/genética , Células Tumorais Cultivadas
4.
J Biol Chem ; 292(29): 12041-12053, 2017 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-28546427

RESUMO

Ubiquitous polyamine spermidine is not required for normal planktonic growth of Bacillus subtilis but is essential for robust biofilm formation. However, the structural features of spermidine required for B. subtilis biofilm formation are unknown and so are the molecular mechanisms of spermidine-stimulated biofilm development. We report here that in a spermidine-deficient B. subtilis mutant, the structural analogue norspermidine, but not homospermidine, restored biofilm formation. Intracellular biosynthesis of another spermidine analogue, aminopropylcadaverine, from exogenously supplied homoagmatine also restored biofilm formation. The differential ability of C-methylated spermidine analogues to functionally replace spermidine in biofilm formation indicated that the aminopropyl moiety of spermidine is more sensitive to C-methylation, which it is essential for biofilm formation, but that the length and symmetry of the molecule is not critical. Transcriptomic analysis of a spermidine-depleted B. subtilis speD mutant uncovered a nitrogen-, methionine-, and S-adenosylmethionine-sufficiency response, resulting in repression of gene expression related to purine catabolism, methionine and S-adenosylmethionine biosynthesis and methionine salvage, and signs of altered membrane status. Consistent with the spermidine requirement in biofilm formation, single-cell analysis of this mutant indicated reduced expression of the operons for production of the exopolysaccharide and TasA protein biofilm matrix components and SinR antagonist slrR Deletion of sinR or ectopic expression of slrR in the spermidine-deficient ΔspeD background restored biofilm formation, indicating that spermidine is required for expression of the biofilm regulator slrR Our results indicate that spermidine functions in biofilm development by activating transcription of the biofilm matrix exopolysaccharide and TasA operons through the regulator slrR.


Assuntos
Bacillus subtilis/fisiologia , Proteínas de Bactérias/agonistas , Biofilmes/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Polissacarídeos Bacterianos/biossíntese , Espermidina/metabolismo , Fatores de Transcrição/agonistas , Adenosilmetionina Descarboxilase/genética , Adenosilmetionina Descarboxilase/metabolismo , Bacillus subtilis/citologia , Bacillus subtilis/genética , Bacillus subtilis/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cadaverina/análogos & derivados , Cadaverina/metabolismo , Deleção de Genes , Perfilação da Expressão Gênica , Metionina/metabolismo , Metilação , Ciclo do Nitrogênio , Óperon , Purinas/metabolismo , S-Adenosilmetionina/metabolismo , Análise de Célula Única , Espermidina/análogos & derivados , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
5.
Biochem J ; 473(10): 1433-41, 2016 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-27001865

RESUMO

Polyamine metabolism is an attractive anticancer drug target, since polyamines are absolutely required for cellular proliferation, and increased levels of polyamines and their biosynthetic enzyme ornithine decarboxylase (ODC) are associated with cancer. Triethylenetetramine (TETA) is a charge-deficient isosteric analogue of the polyamine spermidine (Spd) and a Cu(II)-chelating compound used for the treatment of Wilson's disease, and it has been implicated as a potential anticancer therapeutic drug. In the present study, we studied the effects of TETA in comparison with two other Cu(II)-chelators, D-penicillamine (PA) and tetrathiomolybdate (TTM), on polyamine metabolism in DU145 prostate carcinoma, MCF-7 breast carcinoma and JEG-3 choriocarcinoma cells. TETA induced antizyme, down-regulated ODC and inhibited [(14)C] Spd uptake. Moreover, it completely prevented α-difluoromethylornithine (DFMO)-induced increase in [(14)C] Spd uptake, and inhibited [(14)C] putrescine (Put) uptake and ODC activity in vivo Seven-day treatment of DU145 cells with TETA caused growth cessation by reducing intracellular polyamine levels and suppressing the formation of hypusinated eukaryotic translation initiation factor 5A (eIF5A). TETA or its N-acetylated metabolites also inhibited spermine (Spm), diamine and semicarbazide-sensitive amine oxidases and decreased the level of intracellular reactive oxygen species. Moreover, TETA inhibited the utilization of Put as energy source via the tricarboxylic acid (TCA) cycle, as indicated by decreased production of (14)CO2 from [(14)C] Put. These results indicate that TETA attacks multiple proven anticancer drug targets not attributed to copper chelation, which warrants further studies to reveal its potential in cancer chemoprevention and cure.


Assuntos
Proliferação de Células/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Poliaminas/metabolismo , Trientina/farmacologia , Amina Oxidase (contendo Cobre) , Linhagem Celular Tumoral , Eflornitina/metabolismo , Feminino , Humanos , Células MCF-7 , Masculino , Molibdênio/farmacologia , Penicilamina/metabolismo , Putrescina/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Espermina/metabolismo
6.
EMBO Mol Med ; 15(11): e17833, 2023 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-37702369

RESUMO

Snyder-Robinson syndrome (SRS) results from mutations in spermine synthase (SMS), which converts the polyamine spermidine into spermine. Affecting primarily males, common manifestations of SRS include intellectual disability, osteoporosis, hypotonia, and seizures. Symptom management is the only treatment. Reduced SMS activity causes spermidine accumulation while spermine levels are reduced. The resulting exaggerated spermidine:spermine ratio is a biochemical hallmark of SRS that tends to correlate with symptom severity. Our studies aim to pharmacologically manipulate polyamine metabolism to correct this imbalance as a therapeutic strategy for SRS. Here we report the repurposing of 2-difluoromethylornithine (DFMO), an FDA-approved inhibitor of polyamine biosynthesis, in rebalancing spermidine:spermine ratios in SRS patient cells. Mechanistic in vitro studies demonstrate that, while reducing spermidine biosynthesis, DFMO also stimulates the conversion of spermidine into spermine in hypomorphic SMS cells and induces uptake of exogenous spermine, altogether reducing the aberrant ratios. In a Drosophila SRS model characterized by reduced lifespan, DFMO improves longevity. As nearly all SRS patient mutations are hypomorphic, these studies form a strong foundation for translational studies with significant therapeutic potential.


Assuntos
Poliaminas , Espermidina , Masculino , Humanos , Poliaminas/metabolismo , Espermidina/metabolismo , Espermidina/farmacologia , Espermina/metabolismo , Eflornitina/farmacologia , Eflornitina/uso terapêutico , Espermina Sintase/genética , Espermina Sintase/metabolismo
7.
Biomolecules ; 13(6)2023 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-37371496

RESUMO

The biogenic polyamines, spermidine (Spd) and spermine (Spm), are present at millimolar concentrations in all eukaryotic cells, where they participate in the regulation of vitally important cellular functions. Polyamine analogs and derivatives are a traditional and important instrument for the investigation of the cellular functions of polyamines, enzymes of their metabolism, and the regulation of the biosynthesis of antizyme-a key downregulator of polyamine homeostasis. Here, we describe convenient gram-scale syntheses of a set of C-methylated analogs of Spd. The biochemical properties of these compounds and the possibility for the regulation of their activity by moving a methyl group along the polyamine backbone and by changing the stereochemistry of the chiral center(s) are discussed.


Assuntos
Poliaminas Biogênicas , Espermidina , Poliaminas/metabolismo , Espermina/metabolismo , Homeostase
8.
bioRxiv ; 2023 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-37034775

RESUMO

Snyder-Robinson Syndrome (SRS) is caused by mutations in the spermine synthase (SMS) gene, the enzyme product of which converts the polyamine spermidine into spermine. Affecting primarily males, common manifestations of SRS include intellectual disability, osteoporosis, hypotonic musculature, and seizures, along with other more variable symptoms. Currently, medical management focuses on treating these symptoms without addressing the underlying molecular cause of the disease. Reduced SMS catalytic activity in cells of SRS patients causes the accumulation of spermidine, while spermine levels are reduced. The resulting exaggeration in spermidine-to-spermine ratio is a biochemical hallmark of SRS that tends to correlate with symptom severity in the patient. Our studies aim to pharmacologically manipulate polyamine metabolism to correct this polyamine imbalance and investigate the potential of this approach as a therapeutic strategy for affected individuals. Here we report the use of difluoromethylornithine (DFMO; eflornithine), an FDA-approved inhibitor of polyamine biosynthesis, in re-establishing normal spermidine-to-spermine ratios in SRS patient cells. Through mechanistic studies, we demonstrate that, while reducing spermidine biosynthesis, DFMO also stimulates the conversion of existing spermidine into spermine in cell lines with hypomorphic variants of SMS. Further, DFMO treatment induces a compensatory uptake of exogenous polyamines, including spermine and spermine mimetics, cooperatively reducing spermidine and increasing spermine levels. In a Drosophila SRS model characterized by reduced lifespan, adding DFMO to the feed extended lifespan. As nearly all known SRS patient mutations are hypomorphic, these studies form a foundation for future translational studies with significant therapeutic potential.

9.
Biomolecules ; 13(10)2023 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-37892133

RESUMO

New antibiotics are unquestionably needed to fight the emergence and spread of multidrug-resistant bacteria. To date, antibiotics targeting bacterial central metabolism have been poorly investigated. By determining the minimal inhibitory concentration (MIC) of desmethylphosphinothricin (Glu-γ-PH), an analogue of glutamate with a phosphinic moiety replacing the γ-carboxyl group, we previously showed its promising antibacterial activity on Escherichia coli. Herein, we synthetized and determined the growth inhibition exerted on E. coli by an L-Leu dipeptide derivative of Glu-γ-PH (L-Leu-D,L-Glu-γ-PH). Furthermore, we compared the growth inhibition obtained with this dipeptide with that exerted by the free amino acid, i.e., Glu-γ-PH, and by their phosphonic and non-desmethylated analogues. All the tested compounds were more effective when assayed in a chemically-defined minimal medium. The dipeptide L-Leu-D,L-Glu-γ-PH had a significantly improved antibacterial activity (2 µg/mL), at a concentration between the non-desmethytaled (0.1 µg/mL) and the phosphonic (80 µg/mL) analogues. Also, in Bacillus subtilis, the dipeptide L-Leu-D,L-Glu-γ-PH displayed an activity comparable to that of the antibiotic amoxicillin. This work highlights the antibacterial relevance of the phosphinic pharmacophore and proposes new avenues for the development of novel antimicrobial drugs containing the phosphinic moiety.


Assuntos
Bacillus subtilis , Dipeptídeos , Bacillus subtilis/metabolismo , Dipeptídeos/química , Escherichia coli/metabolismo , Ácido Glutâmico/metabolismo , Antibacterianos/farmacologia , Antibacterianos/metabolismo
10.
Front Pharmacol ; 14: 1327603, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38169863

RESUMO

Oligoarginine peptides, known mostly for their cell-penetrating properties, are also inhibitors of the nicotinic acetylcholine receptors (nAChRs). Since octa-arginine (R8) inhibits α9α10 nAChR and suppresses neuropathic pain, we checked if other polycationic compounds containing amino and/or guanidino groups could be effective and tested the activity of the disulfide-fixed "cyclo"R8, a series of biogenic polyamines (putrescine, spermidine, and spermine), C-methylated spermine analogs, agmatine and its analogs, as well as acylpolyamine argiotoxin-636 from spider venom. Their inhibitory potency on muscle-type, α7 and α9α10 nAChRs was determined using radioligand analysis, electrophysiology, and calcium imaging. "Cyclo"R8 showed similar activity to that of R8 against α9α10 nAChR (IC50 ≈ 60 nM). Biogenic polyamines as well as agmatine and its analogs displayed low activity on muscle-type Torpedo californica, as well as α7 and α9α10 nAChRs, which increased with chain length, the most active being spermine and its C-methylated derivatives having IC50 of about 30 µM against muscle-type T. californica nAChR. Argiotoxin-636, which contains a polyamine backbone and terminal guanidino group, also weakly inhibited T. californica nAChR (IC50 ≈ 15 µM), but it revealed high potency against rat α9α10 nAChR (IC50 ≈ 200 nM). We conclude that oligoarginines and similar polycationic compounds effectively inhibiting α9α10 nAChR may serve as a basis for the development of analgesics to reduce neuropathic pain.

11.
Amino Acids ; 42(2-3): 685-95, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21861168

RESUMO

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 5A
12.
Materials (Basel) ; 14(21)2021 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-34771957

RESUMO

In the present work, the mechanical properties of the DLD-processed Ti-6Al-4V alloy were obtained by tensile tests performed at different temperatures, ranging from 20 °C to 800 °C. Thereby, the process conditions were close to the conditions used to produce large-sized structures using the DLD method, resulting in specimens having the same initial martensitic microstructure. According to the obtained stress curves, the yield strength decreases gradually by 40% when the temperature is increased to 500 °C. Similar behavior is observed for the tensile strength. However, further heating above 500 °C leads to a significant increase in the softening rate. It was found that the DLD-processed Ti-6Al-4V alloy had a Young's modulus with higher thermal stability than conventionally processed alloys. At 500 °C, the Young's modulus of the DLD alloy was 46% higher than that of the wrought alloy. The influence of the thermal history on the stress relaxation for the cases where 500 °C and 700 °C were the maximum temperatures was studied. It was revealed that stress relaxation processes are decisive for the formation of residual stresses at temperatures above 700 °C, which is especially important for small-sized parts produced by the DLD method. The coefficient of thermal expansion was investigated up to 1050 °C.

13.
Biomolecules ; 11(5)2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-34068700

RESUMO

Polyamine spermidine is essential for the proliferation of eukaryotic cells. Administration of polyamine biosynthesis inhibitor α-difluoromethylornithine (DFMO) induces cytostasis that occurs in two phases; the early phase which can be reversed by spermidine, spermine, and some of their analogs, and the late phase which is characterized by practically complete depletion of cellular spermidine pool. The growth of cells at the late phase can be reversed by spermidine and by very few of its analogs, including (S)-1-methylspermidine. It was reported previously (Witherspoon et al. Cancer Discovery 3(9); 1072-81, 2013) that DFMO treatment leads to depletion of cellular thymidine pools, and that exogenous thymidine supplementation partially prevents DFMO-induced cytostasis without affecting intracellular polyamine pools in HT-29, SW480, and LoVo colorectal cancer cells. Here we show that thymidine did not prevent DFMO-induced cytostasis in DU145, LNCaP, MCF7, CaCo2, BT4C, SV40MES13, HepG2, HEK293, NIH3T3, ARPE19 or HT-29 cell lines, whereas administration of functionally active mimetic of spermidine, (S)-1-methylspermidine, did. Thus, the effect of thymidine seems to be specific only for certain cell lines. We conclude that decreased polyamine levels and possibly also distorted pools of folate-dependent metabolites mediate the anti-proliferative actions of DFMO. However, polyamines are necessary and sufficient to overcome DFMO-induced cytostasis, while thymidine is generally not.


Assuntos
Citostáticos/farmacologia , Eflornitina/efeitos adversos , Neoplasias/tratamento farmacológico , Poliaminas/farmacologia , Timidina/farmacologia , Animais , Células Cultivadas , Humanos , Camundongos , Neoplasias/metabolismo , Neoplasias/patologia , Inibidores da Ornitina Descarboxilase/efeitos adversos , Inibidores da Ornitina Descarboxilase/farmacologia
14.
Amino Acids ; 38(2): 509-17, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19997759

RESUMO

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/farmacologia
15.
ACS Infect Dis ; 6(9): 2490-2501, 2020 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-32687697

RESUMO

Viruses require host cell metabolites to productively infect, and the mechanisms by which viruses usurp these molecules are diverse. One group of cellular metabolites important in virus infection is the polyamines, small positively charged molecules involved in cell cycle, translation, and nucleic acid metabolism, among other cellular functions. Polyamines support replication of diverse viruses, and they are important for processes such as transcription, translation, and viral protein enzymatic activity. Rift Valley fever virus (RVFV) is a negative and ambisense RNA virus that requires polyamines to produce infectious particles. In polyamine depleted conditions, noninfectious particles are produced that interfere with virus replication and stimulate immune signaling. Here, we find that RVFV relies on virion-associated polyamines to maintain infectivity and enhance viral entry. We show that RVFV replication is facilitated by a limited set of polyamines and that spermidine and closely related molecules associate with purified virions and transmit from cell to cell during infection. Virion-associated spermidine maintains virion infectivity, as virions devoid of polyamines rapidly lose infectivity and are temperature sensitive. Further, virions without polyamines bind to cells but exhibit a defect in entry, requiring more acidic conditions than virions containing spermidine. These data highlight a unique role for polyamines, and spermidine particularly, to maintain virus infectivity. Further, these studies are the first to identify polyamines associated with RVFV virions. Targeting polyamines represents a promising antiviral strategy, and this work highlights a new mechanism by which we can inhibit virus replication through FDA-approved polyamine depleting pharmaceuticals.


Assuntos
Poliaminas , Vírus da Febre do Vale do Rift , Animais , Proteínas Virais , Vírion , Replicação Viral
16.
Biomolecules ; 10(3)2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32155745

RESUMO

The biogenic polyamines, spermine, spermidine (Spd) and putrescine (Put) are present at micro-millimolar concentrations in eukaryotic and prokaryotic cells (many prokaryotes have no spermine), participating in the regulation of cellular proliferation and differentiation. In mammalian cells Put is formed exclusively from L-ornithine by ornithine decarboxylase (ODC) and many potent ODC inhibitors are known. In bacteria, plants, and fungi Put is synthesized also from agmatine, which is formed from L-arginine by arginine decarboxylase (ADC). Here we demonstrate that the isosteric hydroxylamine analogue of agmatine (AO-Agm) is a new and very potent (IC50 3•10-8 M) inhibitor of E. coli ADC. It was almost two orders of magnitude less potent towards E. coli ODC. AO-Agm decreased polyamine pools and inhibited the growth of DU145 prostate cancer cells only at high concentration (1 mM). Growth inhibitory analysis of the Acremonium chrysogenum demonstrated that the wild type (WT) strain synthesized Put only from L-ornithine, while the cephalosporin C high-yielding strain, in which the polyamine pool is increased, could use both ODC and ADC to produce Put. Thus, AO-Agm is an important addition to the set of existing inhibitors of the enzymes of polyamine biosynthesis, and an important instrument for investigating polyamine biochemistry.


Assuntos
Acremonium/química , Agmatina , Carboxiliases , Proteínas de Escherichia coli , Escherichia coli/enzimologia , Agmatina/análogos & derivados , Agmatina/química , Animais , Carboxiliases/antagonistas & inibidores , Carboxiliases/química , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/química , Masculino , Camundongos
17.
J Med Chem ; 62(24): 11335-11347, 2019 12 26.
Artigo em Inglês | MEDLINE | ID: mdl-31765147

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 Oxidase
18.
ACS Chem Biol ; 11(2): 491-9, 2016 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-26682642

RESUMO

The ubiquitous polyamine spermidine is indispensable for eukaryotic growth and cell proliferation. A conserved vital function of spermidine across eukaryotes is conferred by its aminobutyl group that is transferred to a single lysine in translation factor eIF5A to form the essential hypusine post-translational modification required for cellular translation. In direct contrast, although spermidine is absolutely essential for growth of α-proteobacterial plant pathogen Agrobacterium tumefaciens, we have found, by employing a suite of natural polyamines and synthetic methylated spermidine analogues together with spermidine biosynthetic mutants, that it is solely the 1,3-diaminopropane moiety of spermidine that is required for growth. Indeed, any polyamine containing an intact terminal 1,3-diaminopropane moiety can replace spermidine for growth, including the simple diamine 1,3-diaminopropane itself, a paradigm shift in understanding polyamine function in bacteria. We have identified for the first time a spermidine retroconversion activity in bacteria, producing diamine putrescine from triamine spermidine; however, exogenously supplied tetraamine spermine is resistant to retroconversion. When spermidine levels are pharmacologically decreased, synthesis of spermine from spermidine is induced via the same biosynthetic enzymes, carboxyspermidine dehydrogenase and carboxyspermidine decarboxylase that produce spermidine from putrescine, the first identification of a spermine biosynthetic pathway in bacteria. This also suggests that spermidine represses spermine biosynthesis, but when spermidine levels decrease, it is then converted by carboxyspermidine dehydrogenase and decarboxylase enzymes to spermine, which is resistant to retroconversion and constitutes a sequestered pool of protected 1,3-diaminopropane modules required for growth. We also identify an efficient N-acetylspermidine deacetylase activity, indicative of a sophisticated bacterial polyamine homeostasis system.


Assuntos
Agrobacterium tumefaciens/crescimento & desenvolvimento , Espermidina/metabolismo , Acetilação , Agrobacterium tumefaciens/química , Agrobacterium tumefaciens/metabolismo , Diaminas/química , Diaminas/metabolismo , Propano/análogos & derivados , Propano/metabolismo , Espermidina/análogos & derivados , Espermidina/química
19.
ACS Chem Biol ; 10(6): 1417-24, 2015 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-25689365

RESUMO

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.


Assuntos
Lisina/análogos & derivados , Fatores de Iniciação de Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Ligação a RNA/metabolismo , Espermidina/metabolismo , Transporte Biológico , Linhagem Celular Tumoral , Eflornitina/farmacologia , Expressão Gênica , Humanos , Lisina/biossíntese , Lisina/metabolismo , Masculino , Inibidores da Ornitina Descarboxilase/farmacologia , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/antagonistas & inibidores , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Fatores de Iniciação de Peptídeos/genética , Proteínas de Ligação a RNA/genética , Espermidina/análogos & derivados , Espermidina/farmacologia , Estereoisomerismo , Especificidade por Substrato , Fator de Iniciação de Tradução Eucariótico 5A
20.
J Med Chem ; 54(13): 4611-8, 2011 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-21639123

RESUMO

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-Atividade
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