RESUMO
The monosaccharide l-Rhamnose is an important component of bacterial cell walls. The first step in the l-rhamnose biosynthetic pathway is catalysed by glucose-1-phosphate thymidylyltransferase (RmlA), which condenses glucose-1-phosphate (Glu-1-P) with deoxythymidine triphosphate (dTTP) to yield dTDP-d-glucose. In addition to the active site where catalysis of this reaction occurs, RmlA has an allosteric site that is important for its function. Building on previous reports, SAR studies have explored further the allosteric site, leading to the identification of very potent P. aeruginosa RmlA inhibitors. Modification at the C6-NH2 of the inhibitor's pyrimidinedione core structure was tolerated. X-ray crystallographic analysis of the complexes of P. aeruginosa RmlA with the novel analogues revealed that C6-aminoalkyl substituents can be used to position a modifiable amine just outside the allosteric pocket. This opens up the possibility of linking a siderophore to this class of inhibitor with the goal of enhancing bacterial cell wall permeability.
Assuntos
Desenho de Fármacos , Nucleotidiltransferases/antagonistas & inibidores , Pirimidinonas/farmacologia , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Modelos Moleculares , Estrutura Molecular , Nucleotidiltransferases/metabolismo , Pseudomonas aeruginosa/enzimologia , Pirimidinonas/síntese química , Pirimidinonas/química , Relação Estrutura-AtividadeRESUMO
The life cycles of apicomplexan parasites progress in accordance with fluxes in cytosolic Ca(2+) Such fluxes are necessary for events like motility and egress from host cells. We used genetically encoded Ca(2+) indicators (GCaMPs) to develop a cell-based phenotypic screen for compounds that modulate Ca(2+) signaling in the model apicomplexan Toxoplasma gondii In doing so, we took advantage of the phosphodiesterase inhibitor zaprinast, which we show acts in part through cGMP-dependent protein kinase (protein kinase G; PKG) to raise levels of cytosolic Ca(2+) We define the pool of Ca(2+) regulated by PKG to be a neutral store distinct from the endoplasmic reticulum. Screening a library of 823 ATP mimetics, we identify both inhibitors and enhancers of Ca(2+) signaling. Two such compounds constitute novel PKG inhibitors and prevent zaprinast from increasing cytosolic Ca(2+) The enhancers identified are capable of releasing intracellular Ca(2+) stores independently of zaprinast or PKG. One of these enhancers blocks parasite egress and invasion and shows strong antiparasitic activity against T. gondii The same compound inhibits invasion of the most lethal malaria parasite, Plasmodium falciparum Inhibition of Ca(2+)-related phenotypes in these two apicomplexan parasites suggests that depletion of intracellular Ca(2+) stores by the enhancer may be an effective antiparasitic strategy. These results establish a powerful new strategy for identifying compounds that modulate the essential parasite signaling pathways regulated by Ca(2+), underscoring the importance of these pathways and the therapeutic potential of their inhibition.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Proteínas Quinases Dependentes de GMP Cíclico , Retículo Endoplasmático , Proteínas de Protozoários , Purinonas/farmacologia , Toxoplasma , Proteínas Quinases Dependentes de GMP Cíclico/genética , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Toxoplasma/genética , Toxoplasma/metabolismoRESUMO
UNLABELLED: HIV-1 maturation inhibitors are a novel class of antiretroviral compounds that consist of two structurally distinct chemical classes: betulinic acid derivatives and the pyridone-based compound PF-46396. It is currently believed that both classes act by similar modes of action to generate aberrant noninfectious particles via inhibition of CA-SP1 cleavage during Gag proteolytic processing. In this study, we utilized a series of novel analogues with decreasing similarity to PF-46396 to determine the chemical groups within PF-46396 that contribute to antiviral activity, Gag binding, and the relationship between these essential properties. A spectrum of antiviral activity (active, intermediate, and inactive) was observed across the analogue series with respect to CA-SP1 cleavage and HIV-1 (NL4-3) replication kinetics in Jurkat T cells. We demonstrate that selected inactive analogues are incorporated into wild-type (WT) immature particles and that one inactive analogue is capable of interfering with PF-46396 inhibition of CA-SP1 cleavage. Mutations that confer PF-46396 resistance can impose a defective phenotype on HIV-1 that can be rescued in a compound-dependent manner. Some inactive analogues retained the capacity to rescue PF-46396-dependent mutants (SP1-A3V, SP1-A3T, and CA-P157S), implying that they can also interact with mutant Gag. The structure-activity relationships observed in this study demonstrate that (i) the tert-butyl group is essential for antiviral activity but is not an absolute requirement for Gag binding, (ii) the trifluoromethyl group is optimal but not essential for antiviral activity, and (iii) the 2-aminoindan group is important for antiviral activity and Gag binding but is not essential, as its replacement is tolerated. IMPORTANCE: Combinations of antiretroviral drugs successfully treat HIV/AIDS patients; however, drug resistance problems make the development of new mechanistic drug classes an ongoing priority. HIV-1 maturation inhibitors are novel as they target the Gag protein, specifically by inhibiting CA-SP1 proteolytic cleavage. The lack of high-resolution structural information of the CA-SP1 target in Gag has hindered our understanding of the inhibitor-binding pocket and maturation inhibitor mode of action. Therefore, we utilized analogues of the maturation inhibitor PF-46396 as chemical tools to determine the chemical components of PF-46396 that contribute to antiviral activity and Gag binding and the relationship between these essential properties. This is the first study to report structure-activity relationships of the maturation inhibitor PF-46396. PF-46396 is chemically distinct from betulinic acid-derived maturation inhibitors; therefore, our data provide a foundation of knowledge that will aid our understanding of how structurally distinct maturation inhibitors act by similar modes of action.
Assuntos
Fármacos Anti-HIV/farmacologia , HIV-1/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Fármacos Anti-HIV/química , Farmacorresistência Viral , Humanos , Células Jurkat , Estrutura Molecular , Mutação , Ligação Proteica , Proteólise/efeitos dos fármacos , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacos , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismoRESUMO
The development of fundamentally new approaches for lignin depolymerization is challenged by the complexity of this aromatic biopolymer. While overly simplified model compounds often lack relevance to the chemistry of lignin, the direct use of lignin streams poses significant analytical challenges to methodology development. Ideally, new methods should be tested on model compounds that are complex enough to mirror the structural diversity in lignin but still of sufficiently low molecular weight to enable facile analysis. In this contribution, we present a new class of advanced (ß-O-4)-(ß-5) dilinkage models that are highly realistic representations of a lignin fragment. Together with selected ß-O-4, ß-5, and ß-ß structures, these compounds provide a detailed understanding of the reactivity of various types of lignin linkages in acid catalysis in conjunction with stabilization of reactive intermediates using ethylene glycol. The use of these new models has allowed for identification of novel reaction pathways and intermediates and led to the characterization of new dimeric products in subsequent lignin depolymerization studies. The excellent correlation between model and lignin experiments highlights the relevance of this new class of model compounds for broader use in catalysis studies. Only by understanding the reactivity of the linkages in lignin at this level of detail can fully optimized lignin depolymerization strategies be developed.
Assuntos
Lignina/química , Solventes/química , Acetais/química , Catálise , Dimerização , Formaldeído/química , Concentração de Íons de Hidrogênio , PolimerizaçãoRESUMO
Conversion of lignin into well-defined aromatic chemicals is a highly attractive goal but is often hampered by recondensation of the formed fragments, especially in acidolysis. Here, we describe new strategies that markedly suppress such undesired pathways to result in diverse aromatic compounds previously not systematically targeted from lignin. Model studies established that a catalytic amount of triflic acid is very effective in cleaving the ß-O-4 linkage, most abundant in lignin. An aldehyde product was identified as the main cause of side reactions under cleavage conditions. Capturing this unstable compound by reaction with diols and by in situ catalytic hydrogenation or decarbonylation lead to three distinct groups of aromatic compounds in high yields acetals, ethanol and ethyl aromatics, and methyl aromatics. Notably, the same product groups were obtained when these approaches were successfully extended to lignin. In addition, the formation of higher molecular weight side products was markedly suppressed, indicating that the aldehyde intermediates play a significant role in these processes. The described strategy has the potential to be generally applicable for the production of interesting aromatic compounds from lignin.
RESUMO
Functionalized phenolic monomers have been generated and isolated from an organosolv lignin through a two-step depolymerization process. Chemoselective catalytic oxidation of ß-O-4 linkages promoted by the DDQ/tBuONO/O2 system was achieved in model compounds, including polymeric models and in real lignin. The oxidized ß-O-4 linkages were then cleaved on reaction with zinc. Compared to many existing methods, this protocol, which can be achieved in one pot, is highly selective, giving rise to a simple mixture of products that can be readily purified to give pure compounds. The functionality present in these products makes them potentially valuable building blocks.
Assuntos
Lignina/química , Fenóis/química , Benzoquinonas/química , Oxidantes/química , Oxirredução , Fenóis/isolamento & purificação , PolimerizaçãoRESUMO
The yeast three-hybrid (Y3H) approach shows considerable promise for the unbiased identification of novel small molecule-protein interactions. In recent years, it has been successfully used to link a number of bioactive molecules to novel protein binding partners. However despite its potential importance as a protein target identification method, the Y3H technique has not yet been widely adopted, in part due to the challenges associated with the synthesis of the complex chemical inducers of dimerisation (CIDs). The development of a modular approach using potentially "off the shelf" synthetic components was achieved and allowed the synthesis of a family of four triazole-containing CIDs, MTX-Cmpd2.2-2.5. These CIDs were then compared using the Y3H approach with three of them giving a strong positive interaction with a known target of compound 2, TgCDPK1. These results showed that the modular nature of our synthetic strategy may help to overcome the challenges currently encountered with CID synthesis and should contribute to the Y3H approach reaching its full potential as an unbiased target identification strategy.
Assuntos
Triazóis/síntese química , Química Click , Avaliação Pré-Clínica de Medicamentos , Proteínas Quinases/biossíntese , Multimerização Proteica/efeitos dos fármacos , Proteínas Recombinantes de Fusão/biossíntese , Toxoplasma/enzimologia , Triazóis/farmacologia , Técnicas do Sistema de Duplo-Híbrido , Leveduras/metabolismoRESUMO
The tenovins are small molecule inhibitors of the NAD(+)-dependent family of protein deacetylases known as the sirtuins. There remains considerable interest in inhibitors of this enzyme family due to possible applications in both cancer and neurodegenerative disease therapy. Through the synthesis of novel tenovin analogues, further insights into the structural requirements for activity against the sirtuins in vitro are provided. In addition, the activity of one of the analogues in cells led to an improved understanding of the function of SirT1 in cells.
Assuntos
Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/farmacologia , Sirtuínas/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Benzamidas/síntese química , Benzamidas/química , Benzamidas/farmacologia , Inibidores de Histona Desacetilases/síntese química , Humanos , Ligação de Hidrogênio , Células MCF-7 , Conformação Molecular , Sirtuínas/química , Relação Estrutura-AtividadeRESUMO
Differentiation of the protozoan parasite Toxoplasma gondii into its latent bradyzoite stage is a key event in the parasite's life cycle. Compound 2 is an imidazopyridine that was previously shown to inhibit the parasite lytic cycle, in part through inhibition of parasite cGMP-dependent protein kinase. We show here that Compound 2 can also enhance parasite differentiation, and we use yeast three-hybrid analysis to identify TgBRADIN/GRA24 as a parasite protein that interacts directly or indirectly with the compound. Disruption of the TgBRADIN/GRA24 gene leads to enhanced differentiation of the parasite, and the TgBRADIN/GRA24 knockout parasites show decreased susceptibility to the differentiation-enhancing effects of Compound 2. This study represents the first use of yeast three-hybrid analysis to study small-molecule mechanism of action in any pathogenic microorganism, and it identifies a previously unrecognized inhibitor of differentiation in T. gondii. A better understanding of the proteins and mechanisms regulating T. gondii differentiation will enable new approaches to preventing the establishment of chronic infection in this important human pathogen.
Assuntos
Imidazóis/farmacologia , Proteínas de Protozoários/genética , Pirimidinas/farmacologia , Toxoplasma/genética , Técnicas do Sistema de Duplo-Híbrido , Região 3'-Flanqueadora , Linhagem Celular , Biblioteca Gênica , Genes de Protozoários , Humanos , Imidazóis/síntese química , Imidazóis/química , Estágios do Ciclo de Vida/efeitos dos fármacos , Metotrexato/química , Metotrexato/farmacologia , Fenótipo , Ligação Proteica , Proteínas de Protozoários/metabolismo , Pirimidinas/síntese química , Pirimidinas/química , Toxoplasma/efeitos dos fármacos , Toxoplasma/crescimento & desenvolvimentoRESUMO
Toxoplasma gondii is a parasite of humans and animals, and a model for other apicomplexans including Plasmodium spp., the causative agents of malaria. Despite many advances, manipulating the T. gondii genome remains labor intensive, and is often restricted to lab-adapted strains or lines carrying mutations that enable selection. Here, we use the RNA-guided Cas9 nuclease to efficiently generate knockouts without selection, and to introduce point mutations and epitope tags into the T. gondii genome. These methods will streamline the functional analysis of parasite genes and enable high-throughput engineering of their genomes.
Assuntos
Proteínas Associadas a CRISPR , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Engenharia Genética , Toxoplasma/genética , Antígenos de Protozoários/química , Antígenos de Protozoários/genética , Sequência de Bases , Sistemas CRISPR-Cas , Linhagem Celular , Reparo do DNA por Junção de Extremidades , Ordem dos Genes , Marcação de Genes/métodos , Loci Gênicos , Genoma de Protozoário , Humanos , Dados de Sequência Molecular , Plasmídeos/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Alinhamento de SequênciaRESUMO
Motility of the protozoan parasite Toxoplasma gondii plays an important role in the parasite's life cycle and virulence within animal and human hosts. Motility is driven by a myosin motor complex that is highly conserved across the Phylum Apicomplexa. Two key components of this complex are the class XIV unconventional myosin, TgMyoA, and its associated light chain, TgMLC1. We previously showed that treatment of parasites with a small-molecule inhibitor of T. gondii invasion and motility, tachypleginA, induces an electrophoretic mobility shift of TgMLC1 that is associated with decreased myosin motor activity. However, the direct target(s) of tachypleginA and the molecular basis of the compound-induced TgMLC1 modification were unknown. We show here by "click" chemistry labelling that TgMLC1 is a direct and covalent target of an alkyne-derivatized analogue of tachypleginA. We also show that this analogue can covalently bind to model thiol substrates. The electrophoretic mobility shift induced by another structural analogue, tachypleginA-2, was associated with the formation of a 225.118 Da adduct on S57 and/or C58, and treatment with deuterated tachypleginA-2 confirmed that the adduct was derived from the compound itself. Recombinant TgMLC1 containing a C58S mutation (but not S57A) was refractory to click labelling and no longer exhibited a mobility shift in response to compound treatment, identifying C58 as the site of compound binding on TgMLC1. Finally, a knock-in parasite line expressing the C58S mutation showed decreased sensitivity to compound treatment in a quantitative 3D motility assay. These data strongly support a model in which tachypleginA and its analogues inhibit the motility of T. gondii by binding directly and covalently to C58 of TgMLC1, thereby causing a decrease in the activity of the parasite's myosin motor.