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1.
J Biol Chem ; 292(37): 15225-15239, 2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28726641

RESUMO

Toxoplasma gondii is considered to be one of the most successful intracellular pathogens, because it can reproduce in varied nutritional milieus, encountered in diverse host cell types of essentially any warm-blooded organism. Our earlier work demonstrated that the acute (tachyzoite) stage of T. gondii depends on cooperativity of glucose and glutamine catabolism to meet biosynthetic demands. Either of these two nutrients can sustain the parasite survival; however, what determines the metabolic plasticity has not yet been resolved. Here, we reveal two discrete phosphoenolpyruvate carboxykinase (PEPCK) enzymes in the parasite, one of which resides in the mitochondrion (TgPEPCKmt), whereas the other protein is not expressed in tachyzoites (TgPEPCKnet). Parasites with an intact glycolysis can tolerate genetic deletions of TgPEPCKmt as well as of TgPEPCKnet, indicating their nonessential roles for tachyzoite survival. TgPEPCKnet can also be ablated in a glycolysis-deficient mutant, while TgPEPCKmt is refractory to deletion. Consistent with this, the lytic cycle of a conditional mutant of TgPEPCKmt in the glycolysis-impaired strain was aborted upon induced repression of the mitochondrial isoform, demonstrating its essential role for the glucose-independent survival of parasites. Isotope-resolved metabolomics of the conditional mutant revealed defective flux of glutamine-derived carbon into RNA-bound ribose sugar as well as metabolites associated with gluconeogenesis, entailing a critical nodal role of PEPCKmt in linking catabolism of glucose and glutamine with anabolic pathways. Our data also suggest a homeostatic function ofTgPEPCKmt in cohesive operation of glycolysis and the tricarboxylic acid cycle in a normal glucose-replete milieu. Conversely, we found that the otherwise integrative enzyme pyruvate carboxylase (TgPyC) is dispensable not only in glycolysis-competent but also in glycolysis-deficient tachyzoites despite a mitochondrial localization. Last but not least, the observed physiology of T. gondii tachyzoites appears to phenocopy cancer cells, which holds promise for developing common therapeutics against both threats.


Assuntos
Mitocôndrias/enzimologia , Modelos Biológicos , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Ciclo do Ácido Cítrico , Deleção de Genes , Gluconeogênese , Glucose/metabolismo , Glutamina/metabolismo , Glicólise , Homeostase , Isoenzimas/genética , Isoenzimas/metabolismo , Metabolômica/métodos , Viabilidade Microbiana , Microscopia de Fluorescência , Mitocôndrias/metabolismo , Mutação , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Piruvato Carboxilase/genética , Piruvato Carboxilase/metabolismo , Proteínas Recombinantes de Fusão , Ribose/biossíntese , Toxoplasma/citologia , Toxoplasma/crescimento & desenvolvimento
2.
Parasitol Res ; 114(2): 501-12, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25416330

RESUMO

Potent compounds do not necessarily make the best drugs in the market. Consequently, with the aim to describe tools that may be fundamental for refining the screening of candidates for animal and preclinical studies and further development, molecules of different structural classes synthesized within the frame of a broad screening platform were evaluated for their trypanocidal activities, cytotoxicities against murine macrophages J774.1 and selectivity indices, as well as for their ligand efficiencies and structural chemical properties. To advance into their modes of action, we also describe the morphological and ultrastructural changes exerted by selected members of each compound class on the parasite Trypanosoma brucei. Our data suggest that the potential organelles targeted are either the flagellar pocket (compound 77, N-Arylpyridinium salt; 15, amino acid derivative with piperazine moieties), the endoplasmic reticulum membrane systems (37, bisquaternary bisnaphthalimide; 77, N-Arylpyridinium salt; 68, piperidine derivative), or mitochondria and kinetoplasts (88, N-Arylpyridinium salt; 68, piperidine derivative). Amino acid derivatives with fumaric acid and piperazine moieties (4, 15) weakly inhibiting cysteine proteases seem to preferentially target acidic compartments. Our results suggest that ligand efficiency indices may be helpful to learn about the relationship between potency and chemical characteristics of the compounds. Interestingly, the correlations found between the physico-chemical parameters of the selected compounds and those of commercial molecules that target specific organelles indicate that our rationale might be helpful to drive compound design toward high activities and acceptable pharmacokinetic properties for all compound families.


Assuntos
Fumaratos/farmacologia , Piperazinas/farmacologia , Piperidinas/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Animais , Linhagem Celular , Cisteína Proteases/efeitos dos fármacos , Fumaratos/química , Concentração de Íons de Hidrogênio , Macrófagos/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Organelas/efeitos dos fármacos , Piperazina , Piperazinas/química , Piperidinas/química , Tripanossomicidas/química , Trypanosoma brucei brucei/ultraestrutura
3.
ChemMedChem ; 7(1): 22-31, 2012 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-22113995

RESUMO

For more than 50 years dequalinium chloride has been used successfully as an antiseptic drug and disinfectant, particularly for clinical purposes. Given the success of dequalinium chloride, several series of mono- and bisquaternary ammonium compounds have been designed and reported to have improved antimicrobial activity. Furthermore, many of them exhibit high activity against mycobacteria and protozoa, especially against plasmodia. This review discusses the structure-activity relationships and the modes of action of the various series of (bis)quaternary ammonium compounds.


Assuntos
Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Bactérias/efeitos dos fármacos , Plasmodium/efeitos dos fármacos , Compostos de Amônio Quaternário/química , Compostos de Amônio Quaternário/farmacologia , Animais , Infecções Bacterianas/tratamento farmacológico , Humanos , Malária/tratamento farmacológico , Testes de Sensibilidade Microbiana , Sais/química , Sais/farmacologia , Relação Estrutura-Atividade
4.
Antimicrob Agents Chemother ; 55(1): 311-20, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20937782

RESUMO

Screening of various bisquaternary bisnaphthalimides against a variety of human pathogens revealed one compound, designated MT02, with strong inhibitory effects against Gram-positive bacteria. The MICs ranged from 0.31 µg/ml against community-acquired methicillin-resistant Staphylococcus aureus (MRSA) lineage USA300 to 20 µg/ml against Streptococcus pneumoniae. Radioactive whole-cell labeling experiments indicated a strong impact of MT02 on bacterial DNA replication. DNA microarray studies generated a transcriptional signature characterized by stronger expression of genes involved in DNA metabolism, DNA replication, SOS response, and transport of positively charged compounds. Furthermore, surface plasmon resonance and gel retardation experiments demonstrated direct binding of MT02 to DNA in a concentration-dependent, reversible, and non-sequence-specific manner. The data presented suggest that the bisquaternary bisnaphthalimide MT02 exerts anti-Gram-positive activity by binding to DNA and thereby preventing appropriate DNA replication.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/efeitos dos fármacos , Substâncias Intercalantes/farmacologia , Antibacterianos/química , Proteínas de Bactérias/genética , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Substâncias Intercalantes/química , Testes de Sensibilidade Microbiana , Estrutura Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Staphylococcus aureus/genética
5.
Bioorg Med Chem ; 18(9): 2998-3003, 2010 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-20382538

RESUMO

The bisquaternary bisnaphthalimides are a versatile class of compounds being active against the malaria parasite Plasmodium falciparum in the lower nanomolar range of concentration combined with no cytotoxicity. The series of compounds is designed as choline analogues and interfering agents of the phosphatidylcholine biosynthesis. The qualitative analysis of the structure-activity relationships (SAR) revealed the importance of a long methylene middle chain of at least 8 methylene groups between the two bisquaternary naphthalimides or a monoquaternary naphthalimide consisting of a long alkyl chain attached to the positively charged nitrogen atom. Since the SARs are different from reported biscationic antimalarial drugs the mode of action remains to be elucidated.


Assuntos
Antimaláricos/farmacologia , Naftalimidas/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Animais , Linhagem Celular , Macrófagos/ultraestrutura , Estrutura Molecular , Plasmodium falciparum/ultraestrutura , Compostos de Amônio Quaternário/farmacologia , Relação Estrutura-Atividade
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