ABSTRACT
BACKGROUND: Urinary tract infections (UTIs) are among the most common bacterial infections worldwide, often caused by uropathogenic Escherichia coli. Multiple bacterial virulence factors or patient characteristics have been linked separately to progressive, more invasive infections. In this study, we aim to identify pathogen- and patient-specific factors that drive the progression to urosepsis by jointly analysing bacterial and host characteristics. METHODS: We analysed 1076 E. coli strains isolated from 825 clinical cases with UTI and/or bacteraemia by whole-genome sequencing (Illumina). Sequence types (STs) were determined via srst2 and capsule loci via fastKaptive. We compared the isolates from urine and blood to confirm clonality. Furthermore, we performed a bacterial genome-wide association study (bGWAS) (pyseer) using bacteraemia as the primary clinical outcome. Clinical data were collected by an electronic patient chart review. We concurrently analysed the association of the most significant bGWAS hit and important patient characteristics with the clinical endpoint bacteraemia using a generalised linear model (GLM). Finally, we designed qPCR primers and probes to detect papGII-positive E. coli strains and prospectively screened E. coli from urine samples (n = 1657) at two healthcare centres. RESULTS: Our patient cohort had a median age of 75.3 years (range: 18.00-103.1) and was predominantly female (574/825, 69.6%). The bacterial phylogroups B2 (60.6%; 500/825) and D (16.6%; 137/825), which are associated with extraintestinal infections, represent the majority of the strains in our collection, many of which encode a polysaccharide capsule (63.4%; 525/825). The most frequently observed STs were ST131 (12.7%; 105/825), ST69 (11.0%; 91/825), and ST73 (10.2%; 84/825). Of interest, in 12.3% (13/106) of cases, the E. coli pairs in urine and blood were only distantly related. In line with previous bGWAS studies, we identified the gene papGII (p-value < 0.001), which encodes the adhesin subunit of the E. coli P-pilus, to be associated with 'bacteraemia' in our bGWAS. In our GLM, correcting for patient characteristics, papGII remained highly significant (odds ratio = 5.27, 95% confidence interval = [3.48, 7.97], p-value < 0.001). An independent cohort of cases which we screened for papGII-carrying E. coli at two healthcare centres further confirmed the increased relative frequency of papGII-positive strains causing invasive infection, compared to papGII-negative strains (p-value = 0.033, chi-squared test). CONCLUSIONS: This study builds on previous work linking papGII with invasive infection by showing that it is a major risk factor for progression from UTI to bacteraemia that has diagnostic potential.
Subject(s)
Bacteremia , Escherichia coli Infections , Sepsis , Urinary Tract Infections , Uropathogenic Escherichia coli , Humans , Female , Adolescent , Young Adult , Adult , Middle Aged , Aged , Aged, 80 and over , Male , Genome-Wide Association Study , Escherichia coli Infections/diagnosis , Urinary Tract Infections/diagnosis , Urinary Tract Infections/microbiology , Risk Factors , Virulence Factors/genetics , Uropathogenic Escherichia coli/genetics , Anti-Bacterial AgentsABSTRACT
Age-related neurodegenerative diseases (NDDs) are associated with the aggregation and propagation of specific pathogenic protein species (e.g., Aß, α-synuclein). However, whether disruption of synaptic homeostasis results from protein misfolding per se rather than accumulation of a specific rogue protein is an unexplored question. Here, we show that error-prone translation, with its frequent outcome of random protein misfolding, is sufficient to recapitulate many early features of NDDs, including perturbed Ca2+ signaling, neuronal hyperexcitability, and mitochondrial dysfunction. Mice expressing the ribosomal ambiguity mutation Rps9 D95N exhibited disrupted synaptic homeostasis resulting in behavioral changes reminiscent of early Alzheimer disease (AD), such as learning and memory deficits, maladaptive emotional responses, epileptiform discharges, suppressed circadian rhythmicity, and sleep fragmentation, accompanied by hippocampal NPY expression and cerebral glucose hypometabolism. Collectively, our findings suggest that random protein misfolding may contribute to the pathogenesis of age-related NDDs, providing an alternative framework for understanding the initiation of AD.
Subject(s)
Alzheimer Disease , Aging , Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Animals , Disease Models, Animal , Glucose , Memory Disorders/metabolism , Mice , Mice, Transgenic , alpha-Synuclein/metabolismABSTRACT
BACKGROUND: Intracellularly active antimicrobial peptides are promising candidates for the development of antibiotics for human applications. However, drug development using peptides is challenging as, owing to their large size, an enormous sequence space is spanned. We built a high-throughput platform that incorporates rapid investigation of the sequence-activity relationship of peptides and enables rational optimization of their antimicrobial activity. The platform is based on deep mutational scanning of DNA-encoded peptides and employs highly parallelized bacterial self-screening coupled to next-generation sequencing as a readout for their antimicrobial activity. As a target, we used Bac71-23, a 23 amino acid residues long variant of bactenecin-7, a potent translational inhibitor and one of the best researched proline-rich antimicrobial peptides. RESULTS: Using the platform, we simultaneously determined the antimicrobial activity of >600,000 Bac71-23 variants and explored their sequence-activity relationship. This dataset guided the design of a focused library of ~160,000 variants and the identification of a lead candidate Bac7PS. Bac7PS showed high activity against multidrug-resistant clinical isolates of E. coli, and its activity was less dependent on SbmA, a transporter commonly used by proline-rich antimicrobial peptides to reach the cytosol and then inhibit translation. Furthermore, Bac7PS displayed strong ribosomal inhibition and low toxicity against eukaryotic cells and demonstrated good efficacy in a murine septicemia model induced by E. coli. CONCLUSION: We demonstrated that the presented platform can be used to establish the sequence-activity relationship of antimicrobial peptides, and showed its usefulness for hit-to-lead identification and optimization of antimicrobial drug candidates.
Subject(s)
Anti-Infective Agents , Escherichia coli , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Anti-Infective Agents/metabolism , Anti-Infective Agents/pharmacology , Antimicrobial Cationic Peptides/chemistry , Antimicrobial Cationic Peptides/genetics , Antimicrobial Cationic Peptides/pharmacology , Antimicrobial Peptides , Escherichia coli/genetics , Escherichia coli/metabolism , Humans , Mice , Microbial Sensitivity Tests , Peptides, Cyclic , Proline/metabolismABSTRACT
We have recently identified point mutation V336Y in mitoribosomal protein Mrps5 (uS5m) as a mitoribosomal ram (ribosomal ambiguity) mutation conferring error-prone mitochondrial protein synthesis. In vivo in transgenic knock-in animals, homologous mutation V338Y was associated with a discrete phenotype including impaired mitochondrial function, anxiety-related behavioral alterations, enhanced susceptibility to noise-induced hearing damage, and accelerated metabolic aging in muscle. To challenge the postulated link between Mrps5 V338Y-mediated misreading and the in vivo phenotype, we introduced mutation G315R into the mouse Mrps5 gene as Mrps5 G315R is homologous to the established bacterial ram mutation RpsE (uS5) G104R. However, in contrast to bacterial translation, the homologous G â R mutation in mitoribosomal Mrps5 did not affect the accuracy of mitochondrial protein synthesis. Importantly, in the absence of mitochondrial misreading, homozygous mutant MrpS5G315R/G315R mice did not show a phenotype distinct from wild-type animals.
Subject(s)
Mitochondrial Proteins , Ribosomal Proteins , Animals , Mice , Mitochondrial Proteins/genetics , Mutation , Phenotype , Phylogeny , Protein Biosynthesis , Ribosomal Proteins/geneticsABSTRACT
The main source of error in gene expression is messenger RNA decoding by the ribosome. Translational accuracy has been suggested on a purely correlative basis to positively coincide with maximum possible life span among different rodent species, but causal evidence that translation errors accelerate aging in vivo and limit life span is lacking. We have now addressed this question experimentally by creating heterozygous knock-in mice that express the ribosomal ambiguity mutation RPS9 D95N, resulting in genome-wide error-prone translation. Here, we show that Rps9 D95N knock-in mice exhibit reduced life span and a premature onset of numerous aging-related phenotypes, such as reduced weight, chest deformation, hunchback posture, poor fur condition, and urinary syndrome, together with lymphopenia, increased levels of reactive oxygen species-inflicted damage, accelerated age-related changes in DNA methylation, and telomere attrition. Our results provide an experimental link between translational accuracy, life span, and aging-related phenotypes in mammals.
Subject(s)
Aging, Premature , Aging/genetics , Aging/metabolism , Aging, Premature/genetics , Animals , Longevity , Mammals/genetics , Mice , Reactive Oxygen Species , TelomereABSTRACT
Proteostasis is a challenge for cellular organisms, as all known protein synthesis machineries are error-prone. Here we show by cell fractionation and microscopy studies that misfolded proteins formed in the endoplasmic reticulum can become associated with and partly transported into mitochondria, resulting in impaired mitochondrial function. Blocking the endoplasmic reticulum-mitochondria encounter structure (ERMES), but not the mitochondrial sorting and assembly machinery (SAM) or the mitochondrial surveillance pathway components Msp1 and Vms1, abrogated mitochondrial sequestration of ER-misfolded proteins. We term this mitochondria-associated proteostatic mechanism for ER-misfolded proteins ERAMS (ER-associated mitochondrial sequestration). We testify to the relevance of this pathway by using mutant α-1-antitrypsin as an example of a human disease-related misfolded ER protein, and we hypothesize that ERAMS plays a role in pathological features such as mitochondrial dysfunction.
Subject(s)
Adenosine Triphosphatases/genetics , Carrier Proteins/genetics , Endoplasmic Reticulum/physiology , Mitochondria/physiology , Protein Folding , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Adenosine Triphosphatases/metabolism , Carrier Proteins/metabolism , HEK293 Cells , Humans , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Random errors in protein synthesis are prevalent and ubiquitous, yet their effect on organismal health has remained enigmatic for over five decades. Here, we studied whether mice carrying the ribosomal ambiguity (ram) mutation Rps2-A226Y, recently shown to increase the inborn error rate of mammalian translation, if at all viable, present any specific, possibly aging-related, phenotype. We introduced Rps2-A226Y using a Cre/loxP strategy. Resulting transgenic mice were mosaic and showed a muscle-related phenotype with reduced grip strength. Analysis of gene expression in skeletal muscle using RNA-Seq revealed transcriptomic changes occurring in an age-dependent manner, involving an interplay of PGC1α, FOXO3, mTOR, and glucocorticoids as key signaling pathways, and finally resulting in activation of a muscle atrophy program. Our results highlight the relevance of translation accuracy, and show how disturbances thereof may contribute to age-related pathologies.
Subject(s)
Muscular Atrophy/genetics , Protein Biosynthesis , Aging , Animals , Female , Male , Mice , Mice, Inbred C57BL , Muscular Atrophy/physiopathology , Mutation , Ribosomes/genetics , TranscriptomeABSTRACT
Mitochondrial misreading, conferred by mutation V338Y in mitoribosomal protein Mrps5, in-vivo is associated with a subtle neurological phenotype. Brain mitochondria of homozygous knock-in mutant Mrps5V338Y/V338Y mice show decreased oxygen consumption and reduced ATP levels. Using a combination of unbiased RNA-Seq with untargeted metabolomics, we here demonstrate a concerted response, which alleviates the impaired functionality of OXPHOS complexes in Mrps5 mutant mice. This concerted response mitigates the age-associated decline in mitochondrial gene expression and compensates for impaired respiration by transcriptional upregulation of OXPHOS components together with anaplerotic replenishment of the TCA cycle (pyruvate, 2-ketoglutarate).
Subject(s)
Aging/metabolism , Brain/metabolism , Gene Expression Regulation , Mitochondria/metabolism , Mitochondrial Proteins/biosynthesis , Mutation, Missense , Protein Biosynthesis , Ribosomal Proteins/biosynthesis , Adenosine Triphosphate/metabolism , Aging/genetics , Aging/pathology , Animals , Brain/pathology , Citric Acid Cycle/genetics , Gene Knock-In Techniques , Mice , Mice, Transgenic , Mitochondria/genetics , Mitochondria/pathology , Mitochondrial Proteins/genetics , Ribosomal Proteins/geneticsABSTRACT
Spectinomycin, an aminocyclitol antibiotic, is subject to inactivation by aminoglycoside modifying enzymes (AMEs) through adenylylation or phosphorylation of the 6-hydroxy group position. In this study, the effects of deoxygenation of the 2- and 6-hydroxy group positions on the spectinomycin actinamine ring are probed to evaluate their relationship to ribosomal binding and the antimicrobial activities of spectinomycin, semisynthetic aminomethyl spectinomycins (amSPCs), and spectinamides. To generate these analogs, an improved synthesis of 6-deoxyspectinomycin was developed using the Barton deoxygenation reaction. 6-Dehydrospectinamide was also synthesized from spectinamide 4 to evaluate the H-bond acceptor character on the C-6 position. All the synthesized analogs were tested for antibacterial activity against a panel of Gram (+) and Gram (-) pathogens, plus Mycobacterium tuberculosis. The molecular contribution of the 2- and 6-hydroxy group and the aryl functionalities of all analogs were examined by measuring inhibition of ribosomal translation and molecular dynamics experiments with MM/GBSA analysis. The results of this work indicate that the 6-hydroxy group, which is the primary target of AMEs, is a required motif for antimicrobial activity in current analogs. Removal of the 6-hydroxy group could be partially rescued by offsetting ribosomal binding contributions made by the aryl side chains found in the spectinamide and amSPCs. This study builds on the knowledge of the structure-activity relationships of spectinomycin analogs and is being used to aid the design of next-generation spectinomycins.
Subject(s)
Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Ribosomes/drug effects , Spectinomycin/chemistry , Anti-Bacterial Agents/chemical synthesis , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Microbial Sensitivity Tests , Molecular Dynamics Simulation , Mycobacterium tuberculosis/chemistry , Mycobacterium tuberculosis/drug effects , Spectinomycin/analogs & derivatives , Structure-Activity RelationshipABSTRACT
A stereoselective synthesis of the ribosome-binding antitumor antibiotic (-)-bactobolin A is reported. The presented approach makes effective use of (-)-quinic acid as a chiral pool starting material and substrate stereocontrol to establish the five contiguous stereocenters of (-)-bactobolin A. The key steps of the synthesis include a stereoselective vinylogous aldol reaction to introduce the unusual dichloromethyl substituent, a completely diastereoselective rhodium(II)-catalyzed C-H amination reaction to set the configuration of the axial amine, and an intramolecular alkoxycarbonylation to build the bicyclic lactone framework. The developed synthetic route was used to prepare 90 mg of (-)-bactobolin A trifluoroacetate in 10% overall yield.
Subject(s)
Benzopyrans/chemical synthesis , Catalysis , StereoisomerismABSTRACT
Apramycin is a structurally unique member of the 2-deoxystreptamine class of aminoglycoside antibiotics characterized by a monosubstituted 2-deoxystreptamine ring that carries an unusual bicyclic eight-carbon dialdose moiety. Because of its unusual structure, apramycin is not susceptible to the most prevalent mechanisms of aminoglycoside resistance including the aminoglycoside-modifying enzymes and the ribosomal methyltransferases whose widespread presence severely compromises all aminoglycosides in current clinical practice. These attributes coupled with minimal ototoxocity in animal models combine to make apramycin an excellent starting point for the development of next-generation aminoglycoside antibiotics for the treatment of multidrug-resistant bacterial infections, particularly the ESKAPE pathogens. With this in mind, we describe the design, synthesis, and evaluation of three series of apramycin derivatives, all functionalized at the 5-position, with the goals of increasing the antibacterial potency without sacrificing selectivity between bacterial and eukaryotic ribosomes and of overcoming the rare aminoglycoside acetyltransferase (3)-IV class of aminoglycoside-modifying enzymes that constitutes the only documented mechanism of antimicrobial resistance to apramycin. We show that several apramycin-5-O-ß-d-ribofuranosides, 5-O-ß-d-eryrthofuranosides, and even simple 5-O-aminoalkyl ethers are effective in this respect through the use of cell-free translation assays with wild-type bacterial and humanized bacterial ribosomes and of extensive antibacterial assays with wild-type and resistant Gram negative bacteria carrying either single or multiple resistance determinants. Ex vivo studies with mouse cochlear explants confirm the low levels of ototoxicity predicted on the basis of selectivity at the target level, while the mouse thigh infection model was used to demonstrate the superiority of an apramycin-5-O-glycoside in reducing the bacterial burden in vivo.
Subject(s)
Aminoacyltransferases/metabolism , Anti-Bacterial Agents/pharmacology , Drug Resistance, Bacterial/drug effects , Glycosides/chemistry , Nebramycin/analogs & derivatives , Anti-Bacterial Agents/chemistry , Carbohydrate Conformation , Carbohydrate Sequence , Ethers/chemistry , Microbial Sensitivity Tests , Nebramycin/chemistry , Nebramycin/pharmacologyABSTRACT
Halogenation of a suitably protected netilmicin derivative enables preparation of 4'-chloro-, bromo-, and iodo derivatives of netilmicin after deprotection. Suzuki coupling of a protected 4'-bromo derivative with phenylboronic acid or butyltrifluoroborate affords the corresponding 4'-phenyl and 4'-butyl derivatives of netilmicin. Sulfenylation of suitably protected netilmicin derivative with ethanesulfenyl chloride followed by deprotection affords 4'-ethylsulfanylnetilmicin. All netilmicin 4'-derivatives displayed reduced levels of inhibition for prokaryotic ribosomes and reduced antibacterial activity against typical Gram-positive and Gram-negative strains. None of the derivatives displayed enhanced target selectivity.
ABSTRACT
Infectious diseases due to multidrug-resistant pathogens, particularly carbapenem-resistant Enterobacteriaceae (CREs), present a major and growing threat to human health and society, providing an urgent need for the development of improved potent antibiotics for their treatment. We describe the design and development of a new class of aminoglycoside antibiotics culminating in the discovery of propylamycin. Propylamycin is a 4'-deoxy-4'-alkyl paromomycin whose alkyl substituent conveys excellent activity against a broad spectrum of ESKAPE pathogens and other Gram-negative infections, including CREs, in the presence of numerous common resistance determinants, be they aminoglycoside modifying enzymes or rRNA methyl transferases. Importantly, propylamycin is demonstrated not to be susceptible to the action of the ArmA resistance determinant whose presence severely compromises the action of plazomicin and all other 4,6-disubstituted 2-deoxystreptamine aminoglycosides. The lack of susceptibility to ArmA, which is frequently encoded on the same plasmid as carbapenemase genes, ensures that propylamycin will not suffer from problems of cross-resistance when used in combination with carbapenems. Cell-free translation assays, quantitative ribosome footprinting, and X-ray crystallography support a model in which propylamycin functions by interference with bacterial protein synthesis. Cell-free translation assays with humanized bacterial ribosomes were used to optimize the selectivity of propylamycin, resulting in reduced ototoxicity in guinea pigs. In mouse thigh and septicemia models of Escherichia coli, propylamycin shows excellent efficacy, which is better than paromomycin. Overall, a simple novel deoxy alkyl modification of a readily available aminoglycoside antibiotic increases the inherent antibacterial activity, effectively combats multiple mechanisms of aminoglycoside resistance, and minimizes one of the major side effects of aminoglycoside therapy.
Subject(s)
Aminoglycosides/chemical synthesis , Aminoglycosides/pharmacology , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/pharmacology , Drug Design , Drug Resistance, Bacterial/drug effects , Enterobacteriaceae/drug effects , Aminoglycosides/chemistry , Animals , Anti-Bacterial Agents/chemistry , Chemistry Techniques, Synthetic , Guinea Pigs , Hexosamines/chemical synthesis , Hexosamines/chemistry , Hexosamines/pharmacology , Hexosamines/toxicity , Mice , Microbial Sensitivity Tests , NIH 3T3 Cells , Structure-Activity RelationshipABSTRACT
The 1555 A to G substitution in mitochondrial 12S A-site rRNA is associated with maternally transmitted deafness of variable penetrance in the absence of otherwise overt disease. Here, we recapitulate the suggested A1555G-mediated pathomechanism in an experimental model of mitoribosomal mistranslation by directed mutagenesis of mitoribosomal protein MRPS5. We first establish that the ratio of cysteine/methionine incorporation and read-through of mtDNA-encoded MT-CO1 protein constitute reliable measures of mitoribosomal misreading. Next, we demonstrate that human HEK293 cells expressing mutant V336Y MRPS5 show increased mitoribosomal mistranslation. As for immortalized lymphocytes of individuals with the pathogenic A1555G mutation, we find little changes in the transcriptome of mutant V336Y MRPS5 HEK cells, except for a coordinated upregulation of transcripts for cytoplasmic ribosomal proteins. Homozygous knock-in mutant Mrps5 V338Y mice show impaired mitochondrial function and a phenotype composed of enhanced susceptibility to noise-induced hearing damage and anxiety-related behavioral alterations. The experimental data in V338Y mutant mice point to a key role of mitochondrial translation and function in stress-related behavioral and physiological adaptations.
Subject(s)
Mitochondria/metabolism , Mitochondrial Proteins/genetics , Ribosomal Proteins/genetics , Aging/genetics , Animals , Behavior, Animal , Brain/cytology , Cysteine/metabolism , Electron Transport Complex IV/genetics , Electron Transport Complex IV/metabolism , Escherichia coli Proteins/genetics , HEK293 Cells , Hearing Disorders/genetics , Humans , Methionine/metabolism , Mice, Transgenic , Mitochondria/genetics , Noise/adverse effects , Protein Biosynthesis , RNA, Messenger , Ribosomes/genetics , Ribosomes/metabolism , Stress, Physiological/geneticsABSTRACT
The preparation of a series of four analogues of the aminoglycoside antibiotics neomycin and paromomycin is described in which ring I, involved in critical binding interactions with the ribosomal target, is replaced by an apramycin-like dioxabicyclo[4.4.0]octane system. The effect of this modification is to lock the hydroxymethyl side chain of the neomycin or paromomycin ring I, as part of the dioxabicyclooctane ring, into either the gauche-gauche or the gauche-trans conformation (respectively, axial or equatorial to the bicyclic system). The antiribosomal activity of these compounds is investigated with cell-free translation assays using both bacterial ribosomes and recombinant hybrid ribosomes carrying eukaryotic decoding A site cassettes. Compounds substituted with an equatorial hydroxyl or amino group in the newly formed ring are considerably more active than their axial diastereomers, lending strong support to crystallographically derived models of aminoglycoside-ribosome interactions. One such bicyclic compound carrying an equatorial hydroxyl group has activity equal to that of the parent yet displays better ribosomal selectivity, predictive of an enhanced therapeutic index. A paromomycin analog lacking the hydroxymethyl ring I side chain is considerably less active than the parent. Antibacterial activity against model Gram negative and Gram positive bacteria is reported for selected compounds, as is activity against ESKAPE pathogens and recombinant bacteria carrying specific resistance determinants. Analogues with a bicyclic ring I carrying equatorial amino or hydroxyl groups mimicking the bound side chains of neomycin and paromomycin, respectively, show excellent activity and, by virtue of their novel structure, retain this activity in strains that are insensitive to the parent compounds.
Subject(s)
Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/chemical synthesis , Nebramycin/analogs & derivatives , Paromomycin/analogs & derivatives , Nebramycin/chemical synthesis , Nebramycin/chemistry , Neomycin/analogs & derivatives , Paromomycin/chemical synthesis , Ribosomes/metabolismABSTRACT
The synthesis of a series of neomycin derivatives carrying the 2-hydroxyethyl substituent on N6' and/or N6â´ both alone and in combination with a 4'-O-ethyl group is described. By means of cell-free translation assays with wild-type bacterial ribosomes and their hybrids with eukaryotic decoding A sites, we investigate how individual substituents and their combinations affect activity and selectivity at the target level. In principle, and as shown by cell-free translation assays, modifications of the N6' and N6â´ positions allow enhancement of target selectivity without compromising antibacterial activity. As with the 6'OH aminoglycoside paromomycin, the 4'-O-ethyl modification affects the ribosomal activity, selectivity, and antibacterial profile of neomycin and its 6'-N-(2-hydroxyethyl) derivatives. The modified aminoglycosides show good antibacterial activity against model Gram-positive and Gram-negative microbes including the ESKAPE pathogens Staphylococcus aureus, Klebsiella pneumoniae, Enterobacter cloacae, and Acinetobacter baumannii.
Subject(s)
Anti-Bacterial Agents/chemical synthesis , Neomycin/analogs & derivatives , Paromomycin/analogs & derivatives , Protein Biosynthesis/drug effects , Ribosomes/drug effects , Acinetobacter baumannii/drug effects , Acinetobacter baumannii/growth & development , Anti-Bacterial Agents/pharmacology , Base Pairing , Base Sequence , Carbohydrate Conformation , Enterobacter cloacae/drug effects , Enterobacter cloacae/growth & development , Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/growth & development , Microbial Sensitivity Tests , Neomycin/pharmacology , Paromomycin/pharmacology , Ribosomes/genetics , Ribosomes/metabolism , Staphylococcus aureus/drug effects , Staphylococcus aureus/growth & development , Structure-Activity Relationship , Subcellular Fractions/drug effects , Subcellular Fractions/metabolismABSTRACT
The antibiotic spectinomycin is a potent inhibitor of bacterial protein synthesis with a unique mechanism of action and an excellent safety index, but it lacks antibacterial activity against most clinically important pathogens. A series of N-benzyl-substituted 3'-(R)-3'-aminomethyl-3'-hydroxy spectinomycins was developed on the basis of a computational analysis of the aminomethyl spectinomycin binding site and structure-guided synthesis. These compounds had ribosomal inhibition values comparable to spectinomycin but showed increased potency against the common respiratory tract pathogens Streptococcus pneumoniae, Haemophilus influenzae, Legionella pneumophila, and Moraxella catarrhalis, as well as the sexually transmitted bacteria Neisseria gonorrhoeae and Chlamydia trachomatis. Non-ribosome-binding 3'-(S) isomers of the lead compounds demonstrated weak inhibitory activity in in vitro protein translation assays and poor antibacterial activity, indicating that the antibacterial activity of the series remains on target against the ribosome. Compounds also demonstrated no mammalian cytotoxicity, improved microsomal stability, and favorable pharmacokinetic properties in rats. The lead compound from the series exhibited excellent chemical stability superior to spectinomycin; no interaction with a panel of human receptors and drug metabolism enzymes, suggesting low potential for adverse reactions or drug-drug interactions in vivo; activity in vitro against a panel of penicillin-, macrolide-, and cephalosporin-resistant S. pneumoniae clinical isolates; and the ability to cure mice of fatal pneumococcal pneumonia and sepsis at a dose of 5 mg/kg. Together, these studies indicate that N-benzyl aminomethyl spectinomycins are suitable for further development to treat drug-resistant respiratory tract and sexually transmitted bacterial infections.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacteria/drug effects , Drug Discovery , Drug Resistance, Bacterial , Respiratory Tract Infections/drug therapy , Sexually Transmitted Diseases, Bacterial/drug therapy , Spectinomycin/pharmacology , Animals , Anti-Bacterial Agents/adverse effects , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/pharmacokinetics , Bacteria/metabolism , Bacteria/pathogenicity , Bacterial Proteins/biosynthesis , Chlorocebus aethiops , Computer Simulation , Computer-Aided Design , Disease Models, Animal , Drug Interactions , Drug Stability , Humans , Male , Mice , Microbial Sensitivity Tests , Models, Molecular , Molecular Structure , Rats , Rats, Sprague-Dawley , Respiratory Tract Infections/diagnosis , Respiratory Tract Infections/microbiology , Ribosomes/drug effects , Ribosomes/metabolism , Sexually Transmitted Diseases, Bacterial/diagnosis , Sexually Transmitted Diseases, Bacterial/microbiology , Spectinomycin/adverse effects , Spectinomycin/analogs & derivatives , Spectinomycin/chemical synthesis , Spectinomycin/pharmacokinetics , Structure-Activity Relationship , Vero CellsABSTRACT
A series of 20 4'-O-glycosides of the aminoglycoside antibiotic paromomycin were synthesized and evaluated for their ability to inhibit protein synthesis by bacterial, mitochondrial and cytosolic ribosomes. Target selectivity, i.e., inhibition of the bacterial ribosome over eukaryotic mitochondrial and cytosolic ribosomes, which is predictive of antibacterial activity with reduced ototoxicity and systemic toxicity, was greater for the equatorial than for the axial pyranosides, and greater for the d-pentopyranosides than for the l-pentopyranosides and d-hexopyranosides. In particular, 4'-O-ß-d-xylopyranosyl paromomycin shows antibacterioribosomal activity comparable to that of paromomycin, but is significantly more selective showing considerably reduced affinity for the cytosolic ribosome and for the A1555G mutant mitochondrial ribosome associated with hypersusceptibility to drug-induced ototoxicity. Compound antibacterioribosomal activity correlates with antibacterial activity, and the ribosomally more active compounds show activity against Escherichia coli, Klebsiella pneumonia, Enterobacter cloacae, Acinetobacter baumannii, and methicillin-resistant Staphylococcus aureus (MRSA). The paromomycin glycosides retain activity against clinical strains of MRSA that are resistant to paromomycin, which is demonstrated to be a consequence of 4'-O-glycosylation blocking the action of 4'-aminoglycoside nucleotidyl transferases by the use of recombinant E. coli carrying the specific resistance determinant.
Subject(s)
Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Bacteria/drug effects , Paromomycin/analogs & derivatives , Paromomycin/pharmacology , Ribosomes/drug effects , Bacteria/cytology , Bacterial Infections/drug therapy , Humans , Molecular ConformationABSTRACT
UNLABELLED: The emerging epidemic of drug resistance places the development of efficacious and safe antibiotics in the spotlight of current research. Here, we report the design of next-generation aminoglycosides. Discovery efforts were driven by rational synthesis focusing on 4' alkylations of the aminoglycoside paromomycin, with the goal to alleviate the most severe and disabling side effect of aminoglycosides-irreversible hearing loss. Compounds were evaluated for target activity in in vitro ribosomal translation assays, antibacterial potency against selected pathogens, cytotoxicity against mammalian cells, and in vivo ototoxicity. The results of this study produced potent compounds with excellent selectivity at the ribosomal target, promising antibacterial activity, and little, if any, ototoxicity upon chronic administration. The favorable biocompatibility profile combined with the promising antibacterial activity emphasizes the potential of next-generation aminoglycosides in the treatment of infectious diseases without the risk of ototoxicity. IMPORTANCE: The ever-widening epidemic of multidrug-resistant infectious diseases and the paucity of novel antibacterial agents emerging from modern screening platforms mandate the reinvestigation of established drugs with an emphasis on improved biocompatibility and overcoming resistance mechanisms. Here, we describe the preparation and evaluation of derivatives of the established aminoglycoside antibiotic paromomycin that effectively remove its biggest deficiency, ototoxicity, and overcome certain bacterial resistance mechanisms.
Subject(s)
Aminoglycosides/pharmacology , Anti-Bacterial Agents/pharmacology , Aminoglycosides/chemical synthesis , Animals , Anti-Bacterial Agents/chemical synthesis , Bacterial Infections/drug therapy , Escherichia coli/drug effects , Guinea Pigs , Hexosamines/chemical synthesis , Hexosamines/pharmacology , Male , Mice , Mice, Inbred ICR , Microbial Sensitivity Tests , NIH 3T3 Cells , Phylogeny , RNA, Ribosomal, 16S/genetics , Ribosomes/drug effects , Staphylococcus aureus/drug effectsABSTRACT
A series of apramycin derivatives was prepared and investigated for antibacterial activity and the ability to inhibit protein synthesis in cell-free translation assays. The effect of various modifications at the 6'- and N7'-positions on antiribosomal activity is discussed in terms of their influence on drug binding to specific residues in the decoding A-site. These studies contribute to the development of a structure-activity relationship for the antibacterial activity of the apramycin class of aminoglycosides and to the future design and development of more active and less toxic antibiotics.