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1.
Bioorg Med Chem Lett ; 110: 129883, 2024 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-39013490

RESUMO

The protozoan parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. are responsible for continued propagation of neglected tropical diseases such as African sleeping sickness, Chagas disease and leishmaniasis respectively. Following a report that captopril targets Leishmania donovani dipeptidyl carboxypeptidase, a series of simple proline amides and captopril analogues were synthesized and found to exhibit 1-2 µM in vitro inhibition and selectivity against Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. The results were corroborated with computational docking studies. Arguably, the synthetic proline amides represent the structurally simplest examples of in vitro pan antiprotozoal compounds.

2.
Cell ; 176(1-2): 306-317.e16, 2019 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-30503212

RESUMO

Trypanosome parasites control their virulence and spread by using quorum sensing (QS) to generate transmissible "stumpy forms" in their host bloodstream. However, the QS signal "stumpy induction factor" (SIF) and its reception mechanism are unknown. Although trypanosomes lack G protein-coupled receptor signaling, we have identified a surface GPR89-family protein that regulates stumpy formation. TbGPR89 is expressed on bloodstream "slender form" trypanosomes, which receive the SIF signal, and when ectopically expressed, TbGPR89 drives stumpy formation in a SIF-pathway-dependent process. Structural modeling of TbGPR89 predicts unexpected similarity to oligopeptide transporters (POT), and when expressed in bacteria, TbGPR89 transports oligopeptides. Conversely, expression of an E. coli POT in trypanosomes drives parasite differentiation, and oligopeptides promote stumpy formation in vitro. Furthermore, the expression of secreted trypanosome oligopeptidases generates a paracrine signal that accelerates stumpy formation in vivo. Peptidase-generated oligopeptide QS signals being received through TbGPR89 provides a mechanism for both trypanosome SIF production and reception.


Assuntos
Proteínas de Membrana Transportadoras/fisiologia , Percepção de Quorum/fisiologia , Trypanosoma/metabolismo , Diferenciação Celular , Sequência Conservada/genética , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Membrana Transportadoras/genética , Oligopeptídeos/genética , Oligopeptídeos/fisiologia , Filogenia , Proteínas de Protozoários/metabolismo , Percepção de Quorum/genética , Transdução de Sinais , Trypanosoma/fisiologia , Trypanosoma brucei brucei/metabolismo , Tripanossomíase Africana/parasitologia , Virulência/fisiologia
3.
Sci Rep ; 6: 33189, 2016 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-27616047

RESUMO

Malaria is a life-threatening disease caused by different species of the protozoan parasite Plasmodium, with P. falciparum being the deadliest. Increasing parasitic resistance to existing antimalarials makes the necessity of novel avenues to treat this disease an urgent priority. The enzymes responsible for the synthesis of phosphatidylcholine and phosphatidylethanolamine are attractive drug targets to treat malaria as their selective inhibition leads to an arrest of the parasite's growth and cures malaria in a mouse model. We present here a detailed study that reveals a mode of action for two P. falciparum choline kinase inhibitors both in vitro and in vivo. The compounds present distinct binding modes to the choline/ethanolamine-binding site of P. falciparum choline kinase, reflecting different types of inhibition. Strikingly, these compounds primarily inhibit the ethanolamine kinase activity of the P. falciparum choline kinase, leading to a severe decrease in the phosphatidylethanolamine levels within P. falciparum, which explains the resulting growth phenotype and the parasites death. These studies provide an understanding of the mode of action, and act as a springboard for continued antimalarial development efforts selectively targeting P. falciparum choline kinase.


Assuntos
Antimaláricos/farmacologia , Colina Quinase/antagonistas & inibidores , Fosfatidiletanolaminas/biossíntese , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/antagonistas & inibidores , Antimaláricos/química , Domínio Catalítico , Células Cultivadas , Colina Quinase/química , Colina Quinase/metabolismo , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Eritrócitos/parasitologia , Humanos , Concentração Inibidora 50 , Cinética , Modelos Moleculares , Plasmodium falciparum/efeitos dos fármacos , Ligação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Trofozoítos/efeitos dos fármacos , Trofozoítos/enzimologia
4.
Parasitology ; 137(9): 1357-92, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20602846

RESUMO

The biological membranes of Trypanosoma brucei contain a complex array of phospholipids that are synthesized de novo from precursors obtained either directly from the host, or as catabolised endocytosed lipids. This paper describes the use of nanoflow electrospray tandem mass spectrometry and high resolution mass spectrometry in both positive and negative ion modes, allowing the identification of approximately 500 individual molecular phospholipids species from total lipid extracts of cultured bloodstream and procyclic form T. brucei. Various molecular species of all of the major subclasses of glycerophospholipids were identified including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol as well as phosphatidic acid, phosphatidylglycerol and cardolipin, and the sphingolipids sphingomyelin, inositol phosphoceramide and ethanolamine phosphoceramide. The lipidomic data obtained in this study will aid future biochemical phenotyping of either genetically or chemically manipulated commonly used bloodstream and procyclic strains of Trypanosoma brucei. Hopefully this will allow a greater understanding of the bizarre world of lipids in this important human pathogen.


Assuntos
Lipídeos/química , Trypanosoma brucei brucei/metabolismo , Tripanossomíase/parasitologia , Humanos , Lipídeos/análise , Espectrometria de Massas , Ácidos Fosfatídicos/análise , Ácidos Fosfatídicos/química , Fosfatidilcolinas/análise , Fosfatidilcolinas/química , Fosfatidiletanolaminas/análise , Fosfatidiletanolaminas/química , Fosfatidilgliceróis/análise , Fosfatidilgliceróis/química , Fosfatidilinositóis/análise , Fosfatidilinositóis/química , Fosfatidilserinas/análise , Fosfatidilserinas/química , Fosfolipídeos/análise , Fosfolipídeos/química , Trypanosoma brucei brucei/química , Trypanosoma brucei brucei/crescimento & desenvolvimento
5.
Org Biomol Chem ; 8(15): 3488-99, 2010 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-20532300

RESUMO

Sugar-nucleotides such as GDP-mannose, GDP-fucose and UDP-glucose are important biomolecules with a central role in carbohydrate and glycoconjugate biosynthesis, metabolism and cell signalling. Analogues and mimics of naturally occurring sugar-nucleotides are sought after as chemical tools and inhibitor candidates for sugar-nucleotide-dependent enzymes including glycosyltransferases. Many sugar-nucleotides bind to their target glycosyltransferases via coordination of the diphosphate group to a divalent metal cofactor in the active site. The identification of uncharged, chemically stable surrogates for the diphosphate group, with the ability to coordinate to a divalent metal, is therefore an important design criteria for the development of sugar-nucleotide mimics. Here, we describe the rational design and synthesis of a novel class of sugar-nucleotide mimics based on a squaryldiamide scaffold, an uncharged phosphate isostere. We demonstrate by comprehensive NMR titration experiments that the new sugar-nucleotide mimics coordinate efficiently to Mg(2+), and provide results from biological studies with a therapeutically relevant mannosyltransferase from Trypanosoma brucei. Our findings suggest that squaryldiamides are a promising template for the development of sugar-nucleotide mimics, and illustrate the considerable potential of the squarylamide group as a fragment for inhibitor design.


Assuntos
Materiais Biomiméticos/química , Ciclobutanos/química , Desenho de Fármacos , Nucleotídeos/química , Sulfonamidas/química , Antiprotozoários/síntese química , Antiprotozoários/química , Antiprotozoários/farmacologia , Materiais Biomiméticos/síntese química , Materiais Biomiméticos/farmacologia , Ciclobutanos/síntese química , Ciclobutanos/farmacologia , Magnésio/química , Espectroscopia de Ressonância Magnética , Manosiltransferases/antagonistas & inibidores , Sulfonamidas/síntese química , Sulfonamidas/farmacologia , Trypanosoma brucei brucei/enzimologia
6.
Biochem J ; 425(3): 603-14, 2010 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-19919534

RESUMO

A putative GDP-Man PP (guanidine diphosphomannose pyrophosphorylase) gene from Trypanosoma brucei (TbGDP-Man PP) was identified in the genome and subsequently cloned, sequenced and recombinantly expressed, and shown to be a catalytically active dimer. Kinetic analysis revealed a Vmax of 0.34 mumol/min per mg of protein and Km values of 67 muM and 12 muM for GTP and mannose 1-phosphate respectively. Further kinetic studies showed GDP-Man was a potent product feedback inhibitor. RNAi (RNA interference) of the cytosolic TbGDP-Man PP showed that mRNA levels were reduced to ~20% of wild-type levels, causing the cells to die after 3-4 days, demonstrating that TbGDP-Man PP is essential in the bloodstream form of T. brucei and thus a potential drug target. The RNAi-induced parasites have a greatly reduced capability to form GDP-Man, leading ultimately to a reduction in their ability to synthesize their essential GPI (glycosylphosphatidylinositol) anchors. The RNAi-induced parasites also showed aberrant N-glycosylation of their major cell-surface glycoprotein, variant surface glycoprotein, with loss of the high-mannose Man9GlcNAc2 N-glycosylation at Asn428 and formation of complex N-glycans at Asn263.


Assuntos
Nucleotidiltransferases/metabolismo , Trypanosoma brucei brucei/metabolismo , Tripanossomíase Africana/sangue , Animais , Catálise , Cromatografia em Gel , Clonagem Molecular , Glicosilação , Humanos , Cinética , Dados de Sequência Molecular , Fenótipo , Interferência de RNA , Proteínas Recombinantes/química , Espectrometria de Massas por Ionização por Electrospray
7.
Bioorg Med Chem Lett ; 19(6): 1749-52, 2009 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-19217283

RESUMO

Drug-like molecules with activity against Trypanosoma brucei are urgently required as potential therapeutics for the treatment of African sleeping sickness. Starting from known inhibitors of other glycosyltransferases, we have developed the first small molecular inhibitors of dolicholphosphate mannose synthase (DPMS), a mannosyltransferase critically involved in glycoconjugate biosynthesis in T. brucei. We show that these DPMS inhibitors prevent the biosynthesis of glycosylphosphatidylinositol (GPI) anchors, and possess trypanocidal activity against live trypanosomes.


Assuntos
Química Farmacêutica/métodos , Manosiltransferases/antagonistas & inibidores , Manosiltransferases/química , Trypanosoma brucei brucei/enzimologia , Tripanossomíase Africana/tratamento farmacológico , Animais , Cristalografia por Raios X/métodos , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Etanol/química , Humanos , Modelos Químicos , Conformação Molecular , Estrutura Molecular
8.
Biochem J ; 381(Pt 2): 405-12, 2004 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-15056070

RESUMO

The reason why Leishmania parasites are susceptible to organic antimonial drugs, the standard chemotherapeutic agents for over 50 years, apparently lies in the fact that the mammalian stage of the parasite reduces the pentavalent form of the administered drug to a trivalent form that causes parasite death. We have identified and characterized a parasite-specific enzyme that can catalyse the reduction of pentavalent antimonials and may therefore be central to the anti-parasite activity of the drug. The unusual protein, a trimer of two-domain monomers in which each domain has some similarity to the Omega class glutathione S-transferases, is a thiol-dependent reductase (designated TDR1) that converts pentavalent antimonials into trivalent antimonials using glutathione as the reductant. The higher abundance of the enzyme in the mammalian stage of the parasite could explain why this parasite form is more susceptible to the drug.


Assuntos
Antiprotozoários/metabolismo , Oxirredutases/metabolismo , Sequência de Aminoácidos , Animais , Antimônio/metabolismo , Gluconato de Antimônio e Sódio/metabolismo , Arsênio/metabolismo , Clonagem Molecular/métodos , Glutarredoxinas , Glutationa Transferase/química , Glutationa Transferase/metabolismo , Humanos , Leishmania major/enzimologia , Leishmania major/crescimento & desenvolvimento , Dados de Sequência Molecular , Oxirredução , Oxirredutases/química , Oxirredutases/genética , Proteína Dissulfeto Redutase (Glutationa)/metabolismo , Proteínas de Protozoários/química , Proteínas Recombinantes/genética , Alinhamento de Sequência/métodos
9.
Phytochemistry ; 61(5): 493-501, 2002 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12409015

RESUMO

Linoleic acid (18:2) is found in a large variety of plant oils but to date there is limited knowledge about the substrate selectivity of acyltransferases required for its incorporation into storage triacylglycerols. We have compared the incorporation of oleoyl (18:1) and linoleoyl (18:2) acyl-CoAs onto lysophosphatidic acid acceptors by sub-cellular fractions prepared from a variety of plant and microbial species. Our assays demonstrated: (1). All lysophosphatidic acid acyltransferase (LPA-AT) enzymes tested incorporated 18:2 acyl groups when presented with an equimolar mix of 18:1 and 18:2 acyl-CoA substrates. The ratio of 18:1 to 18:2 incorporation into phosphatidic acid varied between 0.4 and 1.4, indicating low selectivity between these substrates. (2). The presence of either stearoyl (18:0) or oleoyl (18:1) groups at the sn-1 position of lysophosphatidic acid did not affect the selectivity of incorporation of 18:1 or 18:2 into the sn-2 position of phosphatidic acid. (3). All LPA-AT enzymes tested incorporated the saturated palmitoyl (16:0) acyl group from equimolar mixtures of 16:0- and 18:1-CoA. The ratios of 18:1 to 16:0 incorporation are generally much higher than those of 18:1 to 18:2 incorporation, varying between 2.1 and 8.6. (4). The LPA-AT from oil palm kernel is an exception as 18:1 and 16:0 are utilised at comparable rates. These results show that, in the majority of species examined, there is no correlation between the final sn-2 composition of oil or membrane lipids and the ability of an LPA-AT to use 18:2 as a substrate in in vitro assays.


Assuntos
Aciltransferases/metabolismo , Escherichia coli/enzimologia , Magnoliopsida/enzimologia , Saccharomyces cerevisiae/enzimologia , Aciltransferases/análise , Óleos de Plantas/metabolismo , Especificidade por Substrato , Fatores de Tempo , Triglicerídeos/metabolismo
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