RESUMEN
Delivering cargo molecules across the plasma membrane is critical for biomedical research, and the need to develop molecularly well-defined tags that enable cargo transportation is ever-increasing. We report here a hydrophilic endocytosis-promoting peptide (EPP6) rich in hydroxyl groups with no positive charge. EPP6 can transport a wide array of small-molecule cargos into a diverse panel of animal cells. Mechanistic studies revealed that it entered the cells through a caveolin- and dynamin-dependent endocytosis pathway, mediated by the surface receptor fibrinogen C domain-containing protein 1. After endocytosis, EPP6 trafficked through early and late endosomes within 30 min. Over time, EPP6 partitioned among cytosol, lysosomes, and some long-lived compartments. It also demonstrated prominent transcytosis abilities in both in vitro and in vivo models. Our study proves that positive charge is not an indispensable feature for hydrophilic cell-penetrating peptides and provides a new category of molecularly well-defined delivery tags for biomedical applications.
Asunto(s)
Péptidos de Penetración Celular , Endocitosis , Animales , Endosomas/metabolismo , Péptidos de Penetración Celular/metabolismo , Lisosomas/metabolismo , Interacciones Hidrofóbicas e HidrofílicasRESUMEN
In Streptomyces coelicolor, we identified a para-hydroxybenzoate (PHB) hydroxylase, encoded by gene pobA (SCO3084), which is responsible for conversion of PHB into PCA (protocatechuic acid), a substrate of the ß-ketoadipate pathway which yields intermediates of the Krebs cycle. We also found that the transcription of pobA is induced by PHB and is negatively regulated by the product of SCO3209, which we named PobR. The product of this gene is highly unusual in that it is the apparent fusion of two IclR family transcription factors. Bioinformatic analyses, in vivo transcriptional assays, electrophoretic mobility shift assays (EMSAs), DNase I footprinting, and isothermal calorimetry (ITC) were used to elucidate the regulatory mechanism of PobR. We found that PobR loses its high affinity for DNA (i.e., the pobA operator) in the presence of PHB, the inducer of pobA transcription. PHB binds to PobR with a KD of 5.8 µM. Size-exclusion chromatography revealed that PobR is a dimer in the absence of PHB and a monomer in the presence of PHB. The crystal structure of PobR in complex with PHB showed that only one of the two IclR ligand binding domains was occupied, and defined how the N-terminal ligand binding domain engages the effector ligand.
Asunto(s)
4-Hidroxibenzoato-3-Monooxigenasa/química , Proteínas Bacterianas/química , Regulación Bacteriana de la Expresión Génica , Parabenos/química , Streptomyces coelicolor/metabolismo , Factores de Transcripción/química , 4-Hidroxibenzoato-3-Monooxigenasa/genética , 4-Hidroxibenzoato-3-Monooxigenasa/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Sitios de Unión , Biotransformación , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Hidroxibenzoatos/química , Hidroxibenzoatos/metabolismo , Cinética , Ligandos , Modelos Moleculares , Parabenos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Streptomyces coelicolor/genética , Especificidad por Sustrato , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Caseinolytic peptidase P (ClpP), a double-ring peptidase with 14 subunits, collaborates with ATPases associated with diverse activities (AAA+) partners to execute ATP-dependent protein degradation. Although many ClpP enzymes self-assemble into catalytically active homo-tetradecamers able to cleave small peptides, the Mycobacterium tuberculosis enzyme consists of discrete ClpP1 and ClpP2 heptamers that require a AAA+ partner and protein-substrate delivery or a peptide agonist to stabilize assembly of the active tetradecamer. Here, we show that cyclic acyldepsipeptides (ADEPs) and agonist peptides synergistically activate ClpP1P2 by mimicking AAA+ partners and substrates, respectively, and determine the structure of the activated complex. Our studies establish the basis of heteromeric ClpP1P2 assembly and function, reveal tight coupling between the conformations of each ring, show that ADEPs bind only to one ring but appear to open the axial pores of both rings, provide a foundation for rational drug development, and suggest strategies for studying the roles of individual ClpP1 and ClpP2 rings in Clp-family proteolysis.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/química , Modelos Biológicos , Mycobacterium tuberculosis/enzimología , Péptido Hidrolasas/metabolismo , Subunidades de Proteína/química , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Activación Enzimática , Estabilidad de Enzimas , Péptidos Cíclicos/química , Péptidos Cíclicos/metabolismo , Unión Proteica , Multimerización de Proteína , Subunidades de Proteína/metabolismo , Especificidad por SustratoRESUMEN
UNLABELLED: Pupylation is a posttranslational modification peculiar to actinobacteria wherein proteins are covalently modified with a small protein called the prokaryotic ubiquitin-like protein (Pup). Like ubiquitination in eukaryotes, this phenomenon has been associated with proteasome-mediated protein degradation in mycobacteria. Here, we report studies of pupylation in a streptomycete that is phylogentically related to mycobacteria. We constructed mutants of Streptomyces coelicolor lacking PafA (Pup ligase), the proteasome, and the Pup-proteasome system. We found that these mutants share a high susceptibility to oxidative stress compared to that of the wild-type strain. Remarkably, we found that the pafA null mutant has a sporulation defect not seen in strains lacking the Pup-proteasome system. In proteomics experiments facilitated by an affinity-tagged variant of Pup, we identified 110 pupylated proteins in S. coelicolor strains having and lacking genes encoding the 20S proteasome. Our findings shed new light on this unusual posttranslational modification and its role in Streptomyces physiology. IMPORTANCE: The presence of 20S proteasomes reminiscent of those in eukaryotes and a functional equivalent of ubiquitin, known as the prokaryotic ubiquitin-like protein (Pup), in actinobacteria have motivated reevaluations of protein homeostasis in prokaryotes. Though the Pup-proteasome system has been studied extensively in mycobacteria, it is much less understood in streptomycetes, members of a large genus of actinobacteria known for highly choreographed life cycles in which phases of morphological differentiation, sporulation, and secondary metabolism are often regulated by protein metabolism. Here, we define constituents of the pupylome in Streptomyces coelicolor for the first time and present new evidence that links pupylation and the oxidative stress response in this bacterium. Surprisingly, we found that the Pup ligase has a Pup-independent role in sporulation.
Asunto(s)
Proteínas Bacterianas/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Streptomyces coelicolor/fisiología , Ubiquitinas/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Datos de Secuencia Molecular , Mutación , Proteómica , Pupa/genética , Pupa/metabolismo , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Ubiquitinas/química , Ubiquitinas/genéticaRESUMEN
The cyclic acyldepsipeptide (ADEP) antibiotics act by binding the ClpP peptidase and dysregulating its activity. Their exocyclic N-acylphenylalanine is thought to structurally mimic the ClpP-binding, (I/L)GF tripeptide loop of the peptidase's accessory ATPases. We found that ADEP analogues with exocyclic N-acyl tripeptides or dipeptides resembling the (I/L)GF motif were weak ClpP activators and had no bioactivity. In contrast, ADEP analogues possessing difluorophenylalanine N-capped with methyl-branched acyl groups-like the side chains of residues in the (I/L)GF motifs-were superior to the parent ADEP with respect to both ClpP activation and bioactivity. We contend that the ADEP's N-acylphenylalanine moiety is not simply a stand-in for the ATPases' (I/L)GF motif; it likely has physicochemical properties that are better suited for ClpP binding. Further, our finding that the methyl-branching on the acyl group of the ADEPs improves activity opens new avenues for optimization.
RESUMEN
Natural products that inhibit the proteasome have been fruitful starting points for the development of drug candidates. Those of the syringolin family have been underexploited in this context. Using the published model for substrate mimicry by the syringolins and knowledge about the substrate preferences of the proteolytic subunits of the human proteasome, we have designed, synthesized, and evaluated syringolin analogs. As some of our analogs inhibit the activity of the proteasome with second-order rate constants 5-fold greater than that of the methyl ester of syringolin B, we conclude that the substrate mimicry model for the syringolins is valid. The improvements in in vitro potency and the activities of particular analogs against leukemia cell lines are strong bases for further development of the syringolins as anti-cancer drugs.
Asunto(s)
Antineoplásicos/química , Péptidos Cíclicos/química , Inhibidores de Proteasoma/química , Antineoplásicos/metabolismo , Antineoplásicos/toxicidad , Productos Biológicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/toxicidad , Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/metabolismo , Inhibidores de Proteasoma/toxicidad , Unión Proteica , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
MarR family proteins constitute a group of >12 000 transcriptional regulators encoded in bacterial and archaeal genomes that control gene expression in metabolism, stress responses, virulence and multi-drug resistance. There is much interest in defining the molecular mechanism by which ligand binding attenuates the DNA-binding activities of these proteins. Here, we describe how PcaV, a MarR family regulator in Streptomyces coelicolor, controls transcription of genes encoding ß-ketoadipate pathway enzymes through its interaction with the pathway substrate, protocatechuate. This transcriptional repressor is the only MarR protein known to regulate this essential pathway for aromatic catabolism. In in vitro assays, protocatechuate and other phenolic compounds disrupt the PcaV-DNA complex. We show that PcaV binds protocatechuate in a 1:1 stoichiometry with the highest affinity of any MarR family member. Moreover, we report structures of PcaV in its apo form and in complex with protocatechuate. We identify an arginine residue that is critical for ligand coordination and demonstrate that it is also required for binding DNA. We propose that interaction of ligand with this arginine residue dictates conformational changes that modulate DNA binding. Our results provide new insights into the molecular mechanism by which ligands attenuate DNA binding in this large family of transcription factors.
Asunto(s)
Proteínas Bacterianas/química , Proteínas de Unión al ADN/química , Proteínas Represoras/química , Streptomyces coelicolor/genética , Arginina/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , ADN Bacteriano/química , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/metabolismo , Hidroxibenzoatos/química , Ligandos , Modelos Moleculares , Regiones Operadoras Genéticas , Fenoles/química , Unión Proteica , Conformación Proteica , Proteínas Represoras/metabolismoRESUMEN
The cyclic acyldepsipeptide (ADEP) antibiotics are a new class of antibacterial agents that kill bacteria via a mechanism that is distinct from all clinically used drugs. These molecules bind and dysregulate the activity of the ClpP peptidase. The potential of these antibiotics as antibacterial drugs has been enhanced by the elimination of pharmacological liabilities through medicinal chemistry efforts. Here, we demonstrate that the ADEP conformation observed in the ADEP-ClpP crystal structure is fortified by transannular hydrogen bonding and can be further stabilized by judicious replacement of constituent amino acids within the peptidolactone core structure with more conformationally constrained counterparts. Evidence supporting constraint of the molecule into the bioactive conformer was obtained by measurements of deuterium-exchange kinetics of hydrogens that were proposed to be engaged in transannular hydrogen bonds. We show that the rigidified ADEP analogs bind and activate ClpP at lower concentrations in vitro. Remarkably, these compounds have up to 1200-fold enhanced antibacterial activity when compared to those with the peptidolactone core structure common to two ADEP natural products. This study compellingly demonstrates how rational modulation of conformational dynamics may be used to improve the bioactivities of natural products.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Depsipéptidos/química , Depsipéptidos/farmacología , Antibacterianos/síntesis química , Cristalografía por Rayos X , Depsipéptidos/síntesis química , Enterococcus faecalis/efectos de los fármacos , Enlace de Hidrógeno , Pruebas de Sensibilidad Microbiana , Conformación Proteica , Staphylococcus aureus/efectos de los fármacos , Streptococcus pneumoniae/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
The development of new antibacterial agents, particularly those with unique biological targets, is essential to keep pace with the inevitable emergence of drug resistance in pathogenic bacteria. We identified the minimal structural component of the cyclic acyldepsipeptide (ADEP) antibiotics that exhibits antibacterial activity. We found that N-acyldifluorophenylalanine fragments function via the same mechanism of action as ADEPs, as evidenced by the requirement of ClpP for the fragments' antibacterial activity, the ability of fragments to activate Bacillus subtilis ClpP in vitro, and the capacity of an N-acyldifluorophenylalanine affinity matrix to capture ClpP from B. subtilis cell lysates. N-acyldifluorophenylalanine fragments are much simpler in structure than the full ADEPs and are also highly amenable to structural diversification. Thus, the stage has been set for the development of non-peptide activators of ClpP that can be used as antibacterial agents.
Asunto(s)
Antibacterianos/farmacología , Bacillus subtilis/efectos de los fármacos , Depsipéptidos/farmacología , Endopeptidasa Clp/antagonistas & inhibidores , Antibacterianos/química , Bacillus subtilis/enzimología , Depsipéptidos/química , Relación Dosis-Respuesta a Droga , Endopeptidasa Clp/química , Endopeptidasa Clp/metabolismo , Activación Enzimática/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Human polyoma- and papillomaviruses are non-enveloped DNA viruses that cause severe pathologies and mortalities. Under circumstances of immunosuppression, JC polyomavirus causes a fatal demyelinating disease called progressive multifocal leukoencephalopathy (PML) and the BK polyomavirus is the etiological agent of polyomavirus-induced nephropathy and hemorrhagic cystitis. Human papillomavirus type 16, another non-enveloped DNA virus, is associated with the development of cancers in tissues like the uterine cervix and oropharynx. Currently, there are no approved drugs or vaccines to treat or prevent polyomavirus infections. We recently discovered that the small molecule Retro-2(cycl), an inhibitor of host retrograde trafficking, blocked infection by several human and monkey polyomaviruses. Here, we report diversity-oriented syntheses of Retro-2(cycl) and evaluation of the resulting analogs using an assay of human cell infections by JC polyomavirus. We defined structure-activity relationships and also discovered analogs with significantly improved potency as suppressors of human polyoma- and papillomavirus infection in vitro. Our findings represent an advance in the development of drug candidates that can broadly protect humans from non-enveloped DNA viruses and toxins that exploit retrograde trafficking as a means for cell entry.
Asunto(s)
Transporte Axonal/efectos de los fármacos , Virus JC/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/virología , Papillomaviridae/efectos de los fármacos , Quinazolinas/farmacología , Internalización del Virus/efectos de los fármacos , Línea Celular , Relación Dosis-Respuesta a Droga , Humanos , Virus JC/fisiología , Estructura Molecular , Papillomaviridae/fisiología , Quinazolinas/síntesis química , Quinazolinas/química , Relación Estructura-ActividadRESUMEN
Proteolytic complexes in Mycobacterium tuberculosis (Mtb), the deadliest bacterial pathogen, are major foci in tuberculosis drug development programs. The Clp proteases, which are essential for Mtb viability, are high-priority targets. These proteases function through the collaboration of ClpP1P2, a barrel-shaped heteromeric peptidase, with associated ATP-dependent chaperones like ClpX and ClpC1 that recognize and unfold specific substrates in an ATP-dependent fashion. The critical interaction of the peptidase and its unfoldase partners is blocked by the competitive binding of acyldepsipeptide antibiotics (ADEPs) to the interfaces of the ClpP2 subunits. The resulting inhibition of Clp protease activity is lethal to Mtb. Here, we report the surprising discovery that a fragment of the ADEPs retains anti-Mtb activity yet stimulates rather than inhibits the ClpXP1P2-catalyzed degradation of proteins. Our data further suggest that the fragment stabilizes the ClpXP1P2 complex and binds ClpP1P2 in a fashion distinct from that of the intact ADEPs. A structure-activity relationship study of the bioactive fragment defines the pharmacophore and points the way toward the development of new drug leads for the treatment of tuberculosis.
Asunto(s)
Antibacterianos , Mycobacterium tuberculosis , Tuberculosis , Humanos , Adenosina Trifosfato/metabolismo , Antibacterianos/farmacología , Antibacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Endopeptidasa Clp/química , Chaperonas Moleculares/metabolismo , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/metabolismo , Péptido Hidrolasas/efectos de los fármacos , Péptido Hidrolasas/metabolismo , Tuberculosis/tratamiento farmacológicoRESUMEN
Each year, fungi cause more than 1.5 billion infections worldwide and have a devastating impact on human health, particularly in immunocompromised individuals or patients in intensive care units. The limited antifungal arsenal and emerging multidrug-resistant species necessitate the development of new therapies. One strategy for combating drug-resistant pathogens is the administration of molecules that restore fungal susceptibility to approved drugs. Accordingly, we carried out a screen to identify small molecules that could restore the susceptibility of pathogenic Candida species to azole antifungals. This screening effort led to the discovery of novel 1,4-benzodiazepines that restore fluconazole susceptibility in resistant isolates of Candida albicans, as evidenced by 100-1,000-fold potentiation of fluconazole activity. This potentiation effect was also observed in azole-tolerant strains of C. albicans and in other pathogenic Candida species. The 1,4-benzodiazepines selectively potentiated different azoles, but not other approved antifungals. A remarkable feature of the potentiation was that the combination of the compounds with fluconazole was fungicidal, whereas fluconazole alone is fungistatic. Interestingly, the potentiators were not toxic to C. albicans in the absence of fluconazole, but inhibited virulence-associated filamentation of the fungus. We found that the combination of the potentiators and fluconazole significantly enhanced host survival in a Galleria mellonella model of systemic fungal infection. Taken together, these observations validate a strategy wherein small molecules can restore the activity of highly used anti-infectives that have lost potency. IMPORTANCE In the last decade, we have been witnessing a higher incidence of fungal infections, due to an expansion of the fungal species capable of causing disease (e.g., Candida auris), as well as increased antifungal drug resistance. Among human fungal pathogens, Candida species are a leading cause of invasive infections and are associated with high mortality rates. Infections by these pathogens are commonly treated with azole antifungals, yet the expansion of drug-resistant isolates has reduced their clinical utility. In this work, we describe the discovery and characterization of small molecules that potentiate fluconazole and restore the susceptibility of azole-resistant and azole-tolerant Candida isolates. Interestingly, the potentiating 1,4-benzodiazepines were not toxic to fungal cells but inhibited their virulence-associated filamentous growth. Furthermore, combinations of the potentiators and fluconazole decreased fungal burdens and enhanced host survival in a Galleria mellonella model of systemic fungal infections. Accordingly, we propose the use of novel antifungal potentiators as a powerful strategy for addressing the growing resistance of fungi to clinically approved drugs.
Asunto(s)
Antifúngicos , Micosis , Humanos , Antifúngicos/farmacología , Antifúngicos/uso terapéutico , Candida , Fluconazol/farmacología , Fluconazol/uso terapéutico , Azoles/farmacología , Preparaciones Farmacéuticas , Pruebas de Sensibilidad Microbiana , Candida albicans , Micosis/tratamiento farmacológico , Farmacorresistencia Fúngica , Benzodiazepinas/farmacología , Benzodiazepinas/uso terapéuticoRESUMEN
We announce the availability of a high-quality draft of the genome sequence of Amycolatopsis sp. strain 39116, one of few bacterial species that are known to consume the lignin component of plant biomass. This genome sequence will further ongoing efforts to use microorganisms for the conversion of plant biomass into fuels and high-value chemicals.
Asunto(s)
Actinobacteria/clasificación , Actinobacteria/genética , Biomasa , Genoma Bacteriano , Plantas , Actinobacteria/metabolismo , Biodegradación Ambiental , Cromosomas Bacterianos , ADN Bacteriano/genética , Regulación Bacteriana de la Expresión Génica , Datos de Secuencia MolecularRESUMEN
We demonstrate that the Ugi reaction enables chemoselective derivatization of biological amines, carboxylic acids, aldehydes, or ketones with a chromophore under one set of reaction conditions, even in the presence of water. Derivatization of neurotransmitters, hormones, disease biomarkers and other metabolites bodes well for systems biology and diagnostic medicine.
Asunto(s)
Aminoácidos/metabolismo , Biomarcadores/química , Cianuros/metabolismo , Metabolómica/métodos , Aminoácidos/química , Cianuros/químicaRESUMEN
We report a concise synthesis of A-factor, the prototypical γ-butyrolactone signalling compound of Streptomyces bacteria. In analogy to enzymatic reactions in A-factor biosynthesis, our synthesis features a tandem esterification-Knoevenagel condensation yielding a 2-acyl butenolide and a surprising, chemoselective conjugate reduction of this α,ß-unsaturated carbonyl compound using sodium cyanoborohydride.
Asunto(s)
4-Butirolactona/química , Streptomyces griseus/química , 4-Butirolactona/biosíntesis , Estructura Molecular , Streptomyces griseus/metabolismoRESUMEN
Isocyanoacetates are uniquely reactive compounds characterized by an ambivalent isocyano functional group and an enolizable α-carbon. It is widely believed that chiral α-substituted isocyanoacetates are configurationally unstable in some synthetically useful isocyanide-based multicomponent reactions. Herein, we demonstrate that chiral isocyanoacetates can be used with minimal to negligible epimerization in a variety of canonical Ugi four-component condensations as well as Joullié-Ugi three-component condensations, reactions that are particularly useful for constructing complex peptide structures in a single synthetic operation.
RESUMEN
Inhibitors of drug efflux pumps have great potential as pharmacological agents that restore the drug susceptibility of multidrug resistant bacterial pathogens. Most attention has been focused on the discovery of small molecules that inhibit the resistance nodulation division (RND) family drug efflux pumps in Gram-negative bacteria. The prototypical inhibitor of RND-family efflux pumps in Gram-negative bacteria is MC-207,110 (Phe-Arg-ß-naphthylamide), a C-capped dipeptide. Here, we report that C-capped dipeptides inhibit two chloramphenicol-specific efflux pumps in Streptomyces coelicolor, a Gram-positive bacterium that is a relative of the human pathogen Mycobacterium tuberculosis. Diversity-oriented synthesis of a library of structurally related C-capped dipeptides via an Ugi four component reaction and screening of the resulting compounds resulted in the discovery of a compound that is threefold more potent as a suppressor of chloramphenicol resistance in S. coelicolor than MC-207,110. Since chloramphenicol resistance in S. coelicolor is mediated by major facilitator superfamily drug efflux pumps, our findings provide the first evidence that C-capped dipeptides can inhibit drug efflux pumps outside of the RND superfamily.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Cloranfenicol/farmacología , Dipéptidos/química , Dipéptidos/farmacología , Proteínas de Transporte de Membrana/metabolismo , Streptomyces coelicolor/efectos de los fármacos , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Humanos , Streptomyces coelicolor/metabolismo , Relación Estructura-ActividadRESUMEN
Clinical manifestations of leishmaniasis range from self-healing, cutaneous lesions to fatal infections of the viscera. With no preventative Leishmania vaccine available, the frontline option against leishmaniasis is chemotherapy. Unfortunately, currently available anti-Leishmania drugs face several obstacles, including toxicity that limits dosing and emergent drug resistant strains in endemic regions. It is, therefore, imperative that more effective drug formulations with decreased toxicity profiles are developed. Previous studies had shown that 2-(((5-Methyl-2-thienyl)methylene)amino)-N-phenylbenzamide (also called Retro-2) has efficacy against Leishmania infections. Structure-activity relationship (SAR) analogs of Retro-2, using the dihydroquinazolinone (DHQZ) base structure, were subsequently described that are more efficacious than Retro-2. However, considering the hydrophobic nature of these compounds that limits their solubility and uptake, the current studies were initiated to determine whether the solubility of Retro-2 and its SAR analogs could be enhanced through encapsulation in amphiphilic polymer nanoparticles. We evaluated encapsulation of these compounds in the amphiphilic, thermoresponsive oligo(ethylene glycol) methacrylate-co-pentafluorostyrene (PFG30) copolymer that forms nanoparticle aggregates upon heating past temperatures of 30°C. The hydrophobic tracer, coumarin 6, was used to evaluate uptake of a hydrophobic molecule into PFG30 aggregates. Mass spectrometry analysis showed considerably greater delivery of encapsulated DHQZ analogs into infected cells and more rapid shrinkage of L. amazonensis communal vacuoles. Moreover, encapsulation in PFG30 augmented the efficacy of Retro-2 and its SAR analogs to clear both L. amazonensis and L. donovani infections. These studies demonstrate that encapsulation of compounds in PFG30 is a viable approach to dramatically increase bioavailability and efficacy of anti-Leishmania compounds.
Asunto(s)
Leishmania , Leishmaniasis , Animales , Disponibilidad Biológica , Leishmaniasis/tratamiento farmacológico , Ratones , Ratones Endogámicos BALB C , PolímerosRESUMEN
cis-Acting RNA elements in the leaders of bacterial mRNA often regulate gene transcription, especially in the context of amino acid metabolism. We determined that the transcription of the auxiliary, antibiotic-resistant tryptophanyl-tRNA synthetase gene (trpRS1) in Streptomyces coelicolor is regulated by a ribosome-mediated attenuator in the 5' leader of its mRNA region. This regulatory element controls gene transcription in response to the physiological effects of indolmycin and chuangxinmycin, two antibiotics that inhibit bacterial tryptophanyl-tRNA synthetases. By mining streptomycete genome sequences, we found several orthologs of trpRS1 that share this regulatory element; we predict that they are regulated in a similar fashion. The validity of this prediction was established through the analysis of a trpRS1 ortholog (SAV4725) in Streptomyces avermitilis. We conclude that the trpRS1 locus is a widely distributed and self-regulating antibiotic resistance cassette. This study provides insights into how auxiliary aminoacyl-tRNA synthetase genes are regulated in bacteria.
Asunto(s)
Antibacterianos/farmacología , Farmacorresistencia Bacteriana/genética , Ribosomas/fisiología , Streptomyces/metabolismo , Transcripción Genética/fisiología , Triptófano-ARNt Ligasa/metabolismo , Secuencia de Bases , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica/fisiología , Datos de Secuencia Molecular , Mutagénesis , Mutación Missense , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Streptomyces/genética , Triptófano-ARNt Ligasa/genéticaRESUMEN
We report that catalytic quantities of the Lewis acidic metal catalysts scandium triflate and bismuth triflate promote conversion of oleic, linoleic, palmitic and myristic acids and their glyceryl triesters to the corresponding methyl esters (biodiesel) in greater than 90% yield upon microwave heating. Additionally, both catalysts could be recovered and reused in esterification reactions at least six times.