RESUMEN
Previous studies have shown that bicyclic azetidines are potent and selective inhibitors of apicomplexan phenylalanine tRNA synthetase (PheRS), leading to parasite growth inhibition in vitro and in vivo, including in models of Toxoplasma infection. Despite these useful properties, additional optimization is required for the development of efficacious treatments of toxoplasmosis from this inhibitor series, in particular, to achieve optimal exposure in the brain. Here, we describe a series of PheRS inhibitors built on a new bicyclic pyrrolidine core scaffold designed to retain the exit-vector geometry of the isomeric bicyclic azetidine core scaffold while offering avenues to sample diverse chemical space. Relative to the parent series, bicyclic pyrrolidines retain reasonable potency and target selectivity for parasite PheRS vs host. Further structure-activity relationship studies revealed that the introduction of aliphatic groups improved potency and ADME and PK properties, including brain exposure. The identification of this new scaffold provides potential opportunities to extend the analogue series to further improve selectivity and potency and ultimately deliver a novel, efficacious treatment of toxoplasmosis.
Asunto(s)
Encéfalo , Fenilalanina-ARNt Ligasa , Pirrolidinas , Toxoplasma , Toxoplasma/efectos de los fármacos , Toxoplasma/enzimología , Pirrolidinas/farmacología , Pirrolidinas/química , Animales , Encéfalo/parasitología , Relación Estructura-Actividad , Fenilalanina-ARNt Ligasa/antagonistas & inhibidores , Fenilalanina-ARNt Ligasa/química , Antiparasitarios/farmacología , Antiparasitarios/química , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Ratones , Toxoplasmosis/tratamiento farmacológico , Humanos , Azetidinas/farmacología , Azetidinas/químicaRESUMEN
Toxoplasma gondii commonly infects humans and while most infections are controlled by the immune response, currently approved drugs are not capable of clearing chronic infection in humans. Hence, approximately one third of the world's human population is at risk of reactivation, potentially leading to severe sequelae. To identify new candidates for treating chronic infection, we investigated a series of compounds derived from diversity-oriented synthesis. Bicyclic azetidines are potent low nanomolar inhibitors of phenylalanine tRNA synthetase (PheRS) in T. gondii, with excellent selectivity. Biochemical and genetic studies validate PheRS as the primary target of bicyclic azetidines in T. gondii, providing a structural basis for rational design of improved analogs. Favorable pharmacokinetic properties of a lead compound provide excellent protection from acute infection and partial protection from chronic infection in an immunocompromised mouse model of toxoplasmosis. Collectively, PheRS inhibitors of the bicyclic azetidine series offer promise for treatment of chronic toxoplasmosis.
Asunto(s)
Antiprotozoarios/administración & dosificación , Azetidinas/administración & dosificación , Inhibidores Enzimáticos/administración & dosificación , Fenilalanina-ARNt Ligasa/antagonistas & inhibidores , Proteínas Protozoarias/antagonistas & inhibidores , Toxoplasma/efectos de los fármacos , Toxoplasma/enzimología , Toxoplasmosis/tratamiento farmacológico , Animales , Antiprotozoarios/química , Azetidinas/química , Inhibidores Enzimáticos/química , Femenino , Humanos , Cinética , Masculino , Ratones , Ratones Endogámicos CBA , Fenilalanina-ARNt Ligasa/química , Fenilalanina-ARNt Ligasa/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Toxoplasma/genética , Toxoplasma/crecimiento & desarrollo , Toxoplasmosis/parasitologíaRESUMEN
Malaria is a parasitic illness caused by the genus Plasmodium from the apicomplexan phylum. Five plasmodial species of P. falciparum (Pf), P. knowlesi, P. malariae, P. ovale, and P. vivax (Pv) are responsible for causing malaria in humans. According to the World Malaria Report 2020, there were 229 million cases and ~ 0.04 million deaths of which 67% were in children below 5 years of age. While more than 3 billion people are at risk of malaria infection globally, antimalarial drugs are their only option for treatment. Antimalarial drug resistance keeps arising periodically and thus threatens the main line of malaria treatment, emphasizing the need to find new alternatives. The availability of whole genomes of P. falciparum and P. vivax has allowed targeting their unexplored plasmodial enzymes for inhibitor development with a focus on multistage targets that are crucial for parasite viability in both the blood and liver stages. Over the past decades, aminoacyl-tRNA synthetases (aaRSs) have been explored as anti-bacterial and anti-fungal drug targets, and more recently (since 2009) aaRSs are also the focus of antimalarial drug targeting. Here, we dissect the structure-based knowledge of the most advanced three aaRSs-lysyl- (KRS), prolyl- (PRS), and phenylalanyl- (FRS) synthetases in terms of development of antimalarial drugs. These examples showcase the promising potential of this family of enzymes to provide druggable targets that stall protein synthesis upon inhibition and thereby kill malaria parasites selectively.
Asunto(s)
Aminoacil-ARNt Sintetasas/química , Antimaláricos/química , Inhibidores Enzimáticos/química , Lisina-ARNt Ligasa/química , Fenilalanina-ARNt Ligasa/química , Plasmodium falciparum/efectos de los fármacos , Proteínas Protozoarias/química , Aminoacil-ARNt Sintetasas/antagonistas & inhibidores , Aminoacil-ARNt Sintetasas/genética , Aminoacil-ARNt Sintetasas/metabolismo , Antimaláricos/farmacología , Dominio Catalítico , Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Expresión Génica , Humanos , Lisina-ARNt Ligasa/antagonistas & inhibidores , Lisina-ARNt Ligasa/genética , Lisina-ARNt Ligasa/metabolismo , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , Modelos Moleculares , Fenilalanina-ARNt Ligasa/antagonistas & inhibidores , Fenilalanina-ARNt Ligasa/genética , Fenilalanina-ARNt Ligasa/metabolismo , Plasmodium falciparum/química , Plasmodium falciparum/enzimología , Plasmodium falciparum/genética , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Mycobacterium tuberculosis (Mtb) remains the deadliest pathogenic bacteria worldwide. The search for new antibiotics to treat drug-sensitive as well as drug-resistant tuberculosis has become a priority. The essential enzyme phenylalanyl-tRNA synthetase (PheRS) is an antibacterial drug target because of the large differences between bacterial and human PheRS counterparts. In a high-throughput screening of 2148 bioactive compounds, PF-3845, which is a known inhibitor of human fatty acid amide hydrolase, was identified inhibiting Mtb PheRS at Ki â¼ 0.73 ± 0.06 µM. The inhibition mechanism was studied with enzyme kinetics, protein structural modeling, and crystallography, in comparison to a PheRS inhibitor of the noted phenyl-thiazolylurea-sulfonamide class. The 2.3-Å crystal structure of Mtb PheRS in complex with PF-3845 revealed its novel binding mode, in which a trifluoromethyl-pyridinylphenyl group occupies the phenylalanine pocket, whereas a piperidine-piperazine urea group binds into the ATP pocket through an interaction network enforced by a sulfate ion. It represents the first non-nucleoside bisubstrate competitive inhibitor of bacterial PheRS. PF-3845 inhibits the in vitro growth of Mtb H37Rv at â¼24 µM, and the potency of PF-3845 increased against an engineered strain Mtb pheS-FDAS, suggesting on target activity in mycobacterial whole cells. PF-3845 does not inhibit human cytoplasmic or mitochondrial PheRS in biochemical assay, which can be explained from the crystal structures. Further medicinal chemistry efforts focused on the piperidine-piperazine urea moiety may result in the identification of a selective antibacterial lead compound.
Asunto(s)
Mycobacterium tuberculosis/enzimología , Fenilalanina-ARNt Ligasa/ultraestructura , Conformación Proteica , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Amidohidrolasas/antagonistas & inhibidores , Amidohidrolasas/química , Secuencia de Aminoácidos/genética , Antibacterianos/química , Sitios de Unión/efectos de los fármacos , Cristalografía por Rayos X , Humanos , Cinética , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/patogenicidad , Fenilalanina-ARNt Ligasa/antagonistas & inhibidores , Fenilalanina-ARNt Ligasa/química , Piperidinas/química , Piperidinas/farmacología , Piridinas/química , Piridinas/farmacología , Tuberculosis Resistente a Múltiples Medicamentos/enzimología , Tuberculosis Resistente a Múltiples Medicamentos/genéticaRESUMEN
The inhibition of Plasmodium cytosolic phenylalanine tRNA-synthetase (cFRS) by a novel series of bicyclic azetidines has shown the potential to prevent malaria transmission, provide prophylaxis, and offer single-dose cure in animal models of malaria. To date, however, the molecular basis of Plasmodium cFRS inhibition by bicyclic azetidines has remained unknown. Here, we present structural and biochemical evidence that bicyclic azetidines are competitive inhibitors of L-Phe, one of three substrates required for the cFRS-catalyzed aminoacylation reaction that underpins protein synthesis in the parasite. Critically, our co-crystal structure of a PvcFRS-BRD1389 complex shows that the bicyclic azetidine ligand binds to two distinct sub-sites within the PvcFRS catalytic site. The ligand occupies the L-Phe site along with an auxiliary cavity and traverses past the ATP binding site. Given that BRD1389 recognition residues are conserved amongst apicomplexan FRSs, this work lays a structural framework for the development of drugs against both Plasmodium and related apicomplexans.
Asunto(s)
Azetidinas/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Malaria/enzimología , Parásitos/enzimología , Fenilalanina-ARNt Ligasa/antagonistas & inhibidores , Fenilalanina-ARNt Ligasa/química , Plasmodium falciparum/enzimología , Secuencia de Aminoácidos , Aminoacilación , Animales , Dominio Catalítico , Citosol/enzimología , Resistencia a Medicamentos/genética , Modelos Moleculares , Mutación/genética , Fenilalanina/metabolismo , Fenilalanina-ARNt Ligasa/metabolismo , Plasmodium falciparum/efectos de los fármacosRESUMEN
Antibiotics to treat drug-resistant Gram-negative infections are urgently needed but challenging to discover. Using a cell-based screen, we identified a simple secondary amine that inhibited the growth of wild-type Escherichia coli and Acinetobacter baumannii but not the growth of the Gram-positive organism Bacillus subtilis. Resistance mutations in E. coli and A. baumannii mapped exclusively to the aminoacyl-tRNA synthetase PheRS. We confirmed biochemically that the compound inhibited PheRS from these organisms and showed that it did not inhibit PheRS from B. subtilis or humans. To understand the basis for the compound's high selectivity for only some PheRS enzymes, we solved crystal structures of E. coli and A. baumannii PheRS complexed with the inhibitor. The structures showed that the compound's benzyl group mimics the benzyl of phenylalanine. The other amine substituent, a 2-(cyclohexen-1-yl)ethyl group, induces a hydrophobic pocket in which it binds. Through bioinformatic analysis and mutagenesis, we show that the ability to induce a complementary hydrophobic pocket that can accommodate the second substituent explains the high selectivity of this remarkably simple molecular scaffold for Gram-negative PheRS. Because this secondary amine scaffold is active against wild-type Gram-negative pathogens but is not cytotoxic to mammalian cells, we suggest that it may be possible to develop it for use in combination antibiotic therapy to treat Gram-negative infections.
Asunto(s)
Aminas/farmacología , Antibacterianos/farmacología , Bacillus subtilis/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Fenilalanina-ARNt Ligasa/antagonistas & inhibidores , Aminas/química , Antibacterianos/química , Bacillus subtilis/enzimología , Inhibidores Enzimáticos/química , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Fenilalanina-ARNt Ligasa/metabolismoRESUMEN
BACKGROUND: Under oxidative stress cytoplasmic aminoacyl-tRNA synthetase (aaRSs) substrate specificity can be compromised, leading to tRNA mischarging and mistranslation of the proteome. Whether similar processes occur in mitochondria, which are major cellular sources of reactive oxygen species (ROS), is unknown. However, relaxed substrate specificity in yeast mitochondrial phenylalanyl-tRNA synthetase (ScmitPheRS) has been reported to increase tRNA mischarging and blocks mitochondrial biogenesis. METHODS: Non-reducing denaturing PAGE, cysteine reactivity studies, MALDI-TOF mass spectrometry, enzyme assay, western blot, growth assay, circular dichroism, dynamic light scattering and fluorescence spectroscopy were used to study the effect of oxidative stress on ScmitPheRS activity. RESULTS: ScmitPheRS is reversibly inactivated under oxidative stress. The targets for oxidative inactivation are two conserved cysteine residues resulting in reversible intra-molecular disulfide bridge formation. Replacement of either conserved cysteine residue increased viability during growth under oxidative stress. CONCLUSION: Formation of intra-molecular disulfide bridge under oxidative stress hinders the tRNAPhe binding of the enzyme, thus inactivating ScmitPheRS reversibly. GENERAL SIGNIFICANCE: The ScmitPheRS activity is compromised under oxidative stress due to formation of intra-molecular disulfide bridge. The sensitivity of ScmitPheRS to oxidation may provide a protective mechanism against error-prone translation under oxidative stress.
Asunto(s)
Mitocondrias/enzimología , Estrés Oxidativo , Fenilalanina-ARNt Ligasa/antagonistas & inhibidores , Fenilalanina-ARNt Ligasa/metabolismo , ARN de Transferencia de Fenilalanina/metabolismo , Saccharomyces cerevisiae/enzimología , Especificidad por SustratoRESUMEN
Antimalarial drugs have thus far been chiefly derived from two sources-natural products and synthetic drug-like compounds. Here we investigate whether antimalarial agents with novel mechanisms of action could be discovered using a diverse collection of synthetic compounds that have three-dimensional features reminiscent of natural products and are underrepresented in typical screening collections. We report the identification of such compounds with both previously reported and undescribed mechanisms of action, including a series of bicyclic azetidines that inhibit a new antimalarial target, phenylalanyl-tRNA synthetase. These molecules are curative in mice at a single, low dose and show activity against all parasite life stages in multiple in vivo efficacy models. Our findings identify bicyclic azetidines with the potential to both cure and prevent transmission of the disease as well as protect at-risk populations with a single oral dose, highlighting the strength of diversity-oriented synthesis in revealing promising therapeutic targets.
Asunto(s)
Antimaláricos/síntesis química , Antimaláricos/farmacología , Azetidinas/uso terapéutico , Descubrimiento de Drogas , Estadios del Ciclo de Vida/efectos de los fármacos , Malaria Falciparum/tratamiento farmacológico , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/crecimiento & desarrollo , Animales , Antimaláricos/administración & dosificación , Antimaláricos/uso terapéutico , Compuestos de Azabiciclo/administración & dosificación , Compuestos de Azabiciclo/síntesis química , Compuestos de Azabiciclo/farmacología , Compuestos de Azabiciclo/uso terapéutico , Azetidinas/administración & dosificación , Azetidinas/efectos adversos , Azetidinas/farmacología , Citosol/enzimología , Modelos Animales de Enfermedad , Femenino , Hígado/efectos de los fármacos , Hígado/parasitología , Macaca mulatta/parasitología , Malaria Falciparum/prevención & control , Malaria Falciparum/transmisión , Masculino , Ratones , Fenilalanina-ARNt Ligasa/antagonistas & inhibidores , Compuestos de Fenilurea/administración & dosificación , Compuestos de Fenilurea/síntesis química , Compuestos de Fenilurea/farmacología , Compuestos de Fenilurea/uso terapéutico , Plasmodium falciparum/citología , Plasmodium falciparum/enzimología , SeguridadRESUMEN
Pseudomonas aeruginosa and Streptococcus pneumoniae are causative agents in a wide range of infections. Genes encoding proteins corresponding to phenylalanyl-tRNA synthetase (PheRS) were cloned from both bacteria. The two forms of PheRS were kinetically evaluated and the K(m)'s for P. aeruginosa PheRS with its three substrates, phenylalanine, ATP and tRNA(Phe) were determined to be 48, 200, and 1.2 µM, respectively, while the K(m)'s for S. pneumoniae PheRS with respect to phenylalanine, ATP and tRNA(Phe) were 21, 225 and 0.94 µM, respectively. P. aeruginosa and S. pneumoniae PheRS were used to screen a natural compound library and a single compound was identified that inhibited the function of both enzymes. The compound inhibited P. aeruginosa and S. pneumoniae PheRS with IC50's of 2.3 and 4.9 µM, respectively. The compound had a K(I) of 0.83 and 0.98 µM against P. aeruginosa and S. pneumoniae PheRS, respectively. The minimum inhibitory concentration (MIC) of the compound was determined against a panel of Gram positive and negative bacteria including efflux pump mutants and hyper-sensitive strains. MICs against wild-type P. aeruginosa and S. pneumoniae cells in culture were determined to be 16 and 32 µg/ml, respectively. The mechanism of action of the compound was determined to be competitive with the amino acid, phenylalanine, and uncompetitive with ATP. There was no inhibition of cytoplasmic protein synthesis, however, partial inhibition of the human mitochondrial PheRS was observed.
Asunto(s)
Productos Biológicos/farmacología , Ensayos Analíticos de Alto Rendimiento , Naftoles/farmacología , Fenilalanina-ARNt Ligasa/antagonistas & inhibidores , Pseudomonas aeruginosa/enzimología , Streptococcus pneumoniae/enzimología , Secuencia de Aminoácidos , Productos Biológicos/análisis , Células Cultivadas , Humanos , Concentración 50 Inhibidora , Pruebas de Sensibilidad Microbiana , Mitocondrias/efectos de los fármacos , Datos de Secuencia Molecular , Fenilalanina-ARNt Ligasa/genética , Fenilalanina-ARNt Ligasa/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , Pseudomonas aeruginosa/genética , Streptococcus pneumoniae/genéticaRESUMEN
Aminoacyl-tRNA synthetases (aa-RS) attracted interest as potential targets for new antibacterial compounds. Most organisms express 20 aa-RSs: one for each amino acid. Aa-RSs are essential proteins in all living organisms. When one aa-RS is inhibited, the corresponding tRNA is not charged and is therefore unavailable for translation. This leads to protein synthesis inhibition, which in turn causes cell growth arrest. Consequently, each compound that inhibits any of the aa-RS could be a potential antibacterial agent. Only one aa-RS inhibitor, the Ile-RS inhibitor mupirocin, is currently marketed as an antibacterial agent. We focused on phenylalanyl (Phe)-tRNA synthetase (Phe-RS), but the described methods are not restricted to Phe-RS and might be adapted to other aa-RS.
Asunto(s)
Aminoacil-ARNt Sintetasas/antagonistas & inhibidores , Bacterias/efectos de los fármacos , Inhibidores Enzimáticos/análisis , Aminoácidos/sangre , Animales , Antibacterianos/análisis , Antibacterianos/farmacología , Bacterias/enzimología , Bacterias/patogenicidad , Sistema Libre de Células , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Ratones , Pruebas de Sensibilidad Microbiana , Fenilalanina-ARNt Ligasa/antagonistas & inhibidores , Sepsis/tratamiento farmacológico , Sepsis/microbiologíaRESUMEN
In this article, we describe for the first time the high-resolution crystal structure of a phenylalanine tRNA synthetase from the pathogenic bacterium Staphylococcus haemolyticus. We demonstrate the subtle yet important structural differences between this enzyme and the previously described Thermus thermophilus ortholog. We also explain the structure-activity relationship of several recently reported inhibitors. The native enzyme crystals were of poor quality--they only diffracted X-rays to 3-5A resolution. Therefore, we have executed a rational surface mutagenesis strategy that has yielded crystals of this 2300-amino acid multidomain protein, diffracting to 2A or better. This methodology is discussed and contrasted with the more traditional domain truncation approach.
Asunto(s)
Proteínas Bacterianas/química , Fenilalanina-ARNt Ligasa/química , Staphylococcus haemolyticus/enzimología , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X/métodos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Mutagénesis , Fenilalanina-ARNt Ligasa/antagonistas & inhibidores , Fenilalanina-ARNt Ligasa/metabolismo , Ingeniería de Proteínas/métodos , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Staphylococcus haemolyticus/genéticaRESUMEN
In the presence of the stable sulfamoyl analogue of phenylalanyl adenylate (Phe-SA), the UUU/UUC sense codon for phenylalanine (Phe) can be silenced and reassigned to a naphthylalanine (Nap) conjugated to tRNAPhe. We have demonstrated the efficiency and selectivity or orthogonality of the Phe-to-Nap reassignment induced by an "orthogonal reacylation stalling" strategy at the single-codon level in the translation of mRNAs of dihydrofolate reductase and a 24-mer oligopeptide. We used a prokaryotic translation system with an essential preincubation, during which the endogenous precharged phenylalanyl-tRNAPhe undergoes deacylation and the reacylation of the resulting tRNAPhe is stalled by the action of Phe-SA to inhibit the phenylalanyl-tRNA synthetase activity. We discuss the significance of the present small-molecule-based approach to sense-codon templated natural-unnatural peptides.
Asunto(s)
Alanina/análogos & derivados , Codón/química , Oligopéptidos/síntesis química , Aminoacil-ARN de Transferencia/química , Acilación , Adenosina/análogos & derivados , Alanina/química , Alanina/genética , Secuencia de Aminoácidos , Codón/genética , Codón/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Silenciador del Gen , Datos de Secuencia Molecular , Oligopéptidos/biosíntesis , Oligopéptidos/genética , Fenilalanina/química , Fenilalanina/genética , Fenilalanina/metabolismo , Fenilalanina-ARNt Ligasa/antagonistas & inhibidores , Fenilalanina-ARNt Ligasa/metabolismo , ARN Mensajero/síntesis química , ARN Mensajero/genética , Aminoacil-ARN de Transferencia/genética , Aminoacil-ARN de Transferencia/metabolismo , Tetrahidrofolato Deshidrogenasa/biosíntesis , Tetrahidrofolato Deshidrogenasa/genéticaRESUMEN
High throughput screening of Staphylococcus aureus phenylalanyl tRNA synthetase (FRS) identified ethanolamine 1 as a sub-micromolar hit. Optimisation studies led to the enantiospecific lead 64, a single-figure nanomolar inhibitor. The inhibitor series shows selectivity with respect to the mammalian enzyme and the potential for broad spectrum bacterial FRS inhibition.
Asunto(s)
Antibacterianos/síntesis química , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Etanolaminas/síntesis química , Etanolaminas/farmacología , Fenilalanina-ARNt Ligasa/antagonistas & inhibidores , Staphylococcus aureus/enzimología , Animales , Antibacterianos/farmacología , Diseño de Fármacos , Cinética , Mamíferos , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Sensibilidad y Especificidad , Staphylococcus aureus/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
In the presence of Phe-SA, the stable sulfamoyl analogue of phenylalanyl adenylate, the codon (UUU/UUC) for phenylalanine (Phe) can be reassigned to naphthylalanine (Nap) bound to tRNA(Phe). The efficiency and selectivity of this Phe-to-Nap reassignment induced by the "orthogonal reacylation stalling" method was demonstrated at the single-codon level in the translation of mRNAs of dihydrofolate reductase (DHFR) and a 24-mer oligopeptide. In the prokaryotic translation system with essential preincubation, the endogenous precharged phenylalanyl-tRNA(Phe) undergoes deacylation and reacylation of the resulting tRNA(Phe) is inhibited by the action of Phe-SA to kill the phenylalanyl-tRNA synthetase activity. The significance of the present small-molecule-based approach to sense-codon templated natural-unnatural peptides is discussed.
Asunto(s)
Adenosina/análogos & derivados , Codón , Inhibidores Enzimáticos/farmacología , Fenilalanina-ARNt Ligasa/antagonistas & inhibidores , Fenilalanina/análogos & derivados , Biosíntesis de Proteínas , Adenosina/química , Adenosina/farmacología , Alanina/análogos & derivados , Alanina/química , Secuencia de Bases , Inhibidores Enzimáticos/química , Datos de Secuencia Molecular , Naftalenos/química , Péptidos/química , Fenilalanina/química , Fenilalanina/farmacología , Ingeniería de Proteínas , ARN de Transferencia de Fenilalanina/química , Moldes Genéticos , Tetrahidrofolato Deshidrogenasa/biosíntesis , Tetrahidrofolato Deshidrogenasa/genéticaRESUMEN
We have identified a series of spirocyclic furan and pyrrolidine inhibitors of Enterococcus faecalis and Staphylococcus aureus phenylalanyl-tRNA synthetases. The most potent analogue 1b showed IC50=5 nM (E. faecalis PheRS) and IC50=2 nM (S. aureus PheRS) with high selectivity over the human enzyme. The crystal X-ray structure of analogue 1b was determined.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Fenilalanina-ARNt Ligasa/antagonistas & inhibidores , ARN Bacteriano/antagonistas & inhibidores , Furanos/química , Furanos/farmacología , Compuestos Heterocíclicos/química , Compuestos Heterocíclicos/farmacología , Fenilalanina-ARNt Ligasa/metabolismo , Pirrolidinas/química , Pirrolidinas/farmacología , ARN Bacteriano/química , ARN Bacteriano/metabolismoRESUMEN
A series of novel heterocyclic analogues have been synthesized and evaluated for their ability to inhibit phenylalanyl-t-RNA synthetases and act as antibacterial agents. Several analogues have good antibacterial activity against Staphylococcus aureus.
Asunto(s)
Antibacterianos/química , Inhibidores Enzimáticos/química , Compuestos Heterocíclicos/química , Fenilalanina-ARNt Ligasa/antagonistas & inhibidores , Antibacterianos/farmacología , Inhibidores Enzimáticos/farmacología , Compuestos Heterocíclicos/farmacología , Pruebas de Sensibilidad Microbiana/estadística & datos numéricos , Fenilalanina-ARNt Ligasa/metabolismoRESUMEN
Ochratoxine A (OTA) inhibits growth of Bacillus subtilis at pHs below 7. Since OTA is a phenylalanine analogue, this effect could be due to inhibition of phenylalanine-tRNA synthetase (PheRS) by competition of this mycotoxin with the amino acid. Homogeneous PheRS was purified from Bacillus subtilis and from E. coli transformed with the PheRS gene. The latter produced about 40 times more PheRS than B. subtilis. The Km and Ki values of PheRS, respectively, for phenylalanine and OTA were measured and their concentrations within the cell determined. It appears that the concentration of OTA in the cell, in spite of a 25-fold accumulation, remained too low to significantly compete with phenylalanine. This does not suggest PheRS to be the target of OTA in cell growth and protein synthesis inhibition in Bacillus subtilis. It was also shown that the 2-3-fold increase of PheRS in OTA-treated cells is not due to phenylalanine-controlled attenuation regulation.
Asunto(s)
Bacillus subtilis/enzimología , Ocratoxinas/farmacología , Fenilalanina-ARNt Ligasa/metabolismo , Bacillus subtilis/efectos de los fármacos , Unión Competitiva , Escherichia coli/enzimología , Escherichia coli/genética , Cinética , Sustancias Macromoleculares , Fenilalanina/metabolismo , Fenilalanina-ARNt Ligasa/antagonistas & inhibidores , Transformación BacterianaRESUMEN
Rapid inactivation of the yeast phenylalanyl-tRNA synthetase in the course of aminoacylation of the heterologous E. coli tRNA(Phe) is observed. This inactivation occurs due to the formation of the tight complex of the enzyme with the pyrophosphate formed during the aminoacylation reaction. This complex is shown to be the normal intermediate of the reaction. Possible inactivation mechanism and correlation between structural differences of yeast and E. coli tRNAs(Phe) with the changes in the enzymatic mechanism of aminoacylation are discussed.
Asunto(s)
Difosfatos/metabolismo , Fenilalanina-ARNt Ligasa/metabolismo , ARN Bacteriano/metabolismo , ARN de Transferencia de Fenilalanina/metabolismo , Acilación , Escherichia coli/metabolismo , Cinética , Fenilalanina-ARNt Ligasa/antagonistas & inhibidores , Levaduras/enzimologíaRESUMEN
Ochratoxin A has a number of toxic effects in mammals, the most notable of which is nephrotoxicity. It is also immunosuppressive, teratogenic and carcinogenic. The biochemical and molecular aspects of its action were first studied in bacteria. The appearance of 'magic spots' (ppGpp and pppGpp) pointed to inhibition of the charging of transfer ribonucleic acids (tRNA) with amino acids. This suggestion was confirmed by the demonstration that ochratoxin A inhibits bacterial, yeast and liver phenylalanyl-tRNA synthetases. The inhibition is competitive to phenylalanine and is reversed by an excess of this amino acid. As a consequence, protein synthesis is inhibited, as shown with hepatoma cells in culture, with Madin Darby canine kidney cells (which are much more sensitive) and in vivo in mouse liver, kidney and spleen, the inhibition being more effective in the latter two organs. An excess of phenylalanine also prevents inhibition of protein synthesis in cell cultures and in vivo. Analogues of ochratoxin A in which phenylalanine has been replaced by other amino acids have similar inhibitory effects on the respective amino acid-specific aminoacyl tRNA synthetases. 4R-Hydroxyochratoxin A, a metabolite of ochratoxin A, has a similar action, whereas ochratoxin alpha (the dihydroisocoumarin moiety) and ochratoxin B (ochratoxin A without chlorine) have no effect. Ochratoxin A might act on other enzymes that use phenylalanine as a substrate. We showed recently that it inhibits phenylalanine hydroxylase. In addition, the phenylalanine moiety of ochratoxin A is partially hydroxylated to tyrosine by incubation with hepatocytes and in vivo. This competitive action with phenylalanine might explain why this amino acid prevents the immuno-suppressive effect of ochratoxin A and partially prevents its teratogenic and nephrotoxic actions. The effect of ochratoxin A on protein synthesis is followed by an inhibition of RNA synthesis, which might affect proteins with a high turnover. Ochratoxin A also lowers the level of phosphoenolpyruvate carboxykinase, a key enzyme in gluconeogenesis; this inhibition is reported to be due to a specific degradation of mRNA that codes for this enzyme. Recently, ochratoxin A was also found to enhance lipid peroxidation both in vitro and in vivo. This inhibition might have an important effect on cell or mitochondrial membranes and be responsible for the effects on mitochondria that have been shown by several authors. Finally, the recent results of Pfohl-Leszkowicz et al. (this volume), who showed the formation of DNA adducts mainly in kidney but also in liver and spleen, explain the DNA single-strand breaks observed previously in mice and rats after acute and chronic treatment.
Asunto(s)
Ocratoxinas/farmacología , Fenilalanina-ARNt Ligasa/antagonistas & inhibidores , Fenilalanina/metabolismo , Animales , Formación de Anticuerpos/efectos de los fármacos , Bacterias/efectos de los fármacos , Células Cultivadas , Daño del ADN , Perros , Células Eucariotas/efectos de los fármacos , Cobayas , Humanos , Túbulos Renales Proximales/efectos de los fármacos , Peroxidación de Lípido/efectos de los fármacos , Neoplasias Hepáticas Experimentales/patología , Ratones , Ratones Endogámicos BALB C , Pruebas de Mutagenicidad , Micotoxicosis/etiología , Ocratoxinas/farmacocinética , Ocratoxinas/toxicidad , Fenilalanina/análogos & derivados , Fenilalanina Hidroxilasa/antagonistas & inhibidores , Fosfoenolpiruvato Carboxiquinasa (GTP)/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , Inhibidores de la Síntesis de la Proteína/toxicidad , Ratas , Relación Estructura-Actividad , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
tRNA(Phe) in which the adenine and cytosine rings in the aminoacyl arm and in the anticodon loop were converted to alkylating derivatives by mild treatment with methyl chlorotetrolate was used to study the tRNA(Phe)-yeast phenylalanyl-tRNA(Phe) synthetase interaction. At neutral pH, modified tRNA inhibited the enzyme competitively. At pH 9 this binding is accompanied by irreversible inactivation of the enzyme due to alkylation of the alpha subunit of the synthetase. Such a derivatization of tRNA could probably be used to investigate the interaction of other tRNAs with their cognate synthetases.