RESUMO
Aminoglycosides are potent antibacterials, but therapy is compromised by substantial toxicity causing, in particular, irreversible hearing loss. Aminoglycoside ototoxicity occurs both in a sporadic dose-dependent and in a genetically predisposed fashion. We recently have developed a mechanistic concept that postulates a key role for the mitochondrial ribosome (mitoribosome) in aminoglycoside ototoxicity. We now report on the surprising finding that apramycin, a structurally unique aminoglycoside licensed for veterinary use, shows little activity toward eukaryotic ribosomes, including hybrid ribosomes which were genetically engineered to carry the mitoribosomal aminoglycoside-susceptibility A1555G allele. In ex vivo cultures of cochlear explants and in the in vivo guinea pig model of chronic ototoxicity, apramycin causes only little hair cell damage and hearing loss but it is a potent antibacterial with good activity against a range of clinical pathogens, including multidrug-resistant Mycobacterium tuberculosis. These data provide proof of concept that antibacterial activity can be dissected from aminoglycoside ototoxicity. Together with 3D structures of apramycin-ribosome complexes at 3.5-Å resolution, our results provide a conceptual framework for further development of less toxic aminoglycosides by hypothesis-driven chemical synthesis.
Assuntos
Aminoglicosídeos/toxicidade , Infecções Bacterianas/tratamento farmacológico , Surdez/induzido quimicamente , Mitocôndrias/efeitos dos fármacos , Nebramicina/análogos & derivados , Ribossomos/efeitos dos fármacos , Animais , Antibacterianos/toxicidade , Bactérias/efeitos dos fármacos , Sítios de Ligação/efeitos dos fármacos , Surdez/fisiopatologia , Desenho de Fármacos , Gentamicinas/toxicidade , Cobaias , Células HEK293 , Células Ciliadas Auditivas/efeitos dos fármacos , Humanos , Camundongos , Mitocôndrias/metabolismo , Mutagênese/fisiologia , Mycobacterium/efeitos dos fármacos , Nebramicina/química , Nebramicina/toxicidade , Técnicas de Cultura de Órgãos , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/fisiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Coelhos , Reticulócitos/citologia , Ribossomos/química , Ribossomos/metabolismo , Staphylococcus aureus/efeitos dos fármacosRESUMO
The kanamycins form an important subgroup of the 4,6-disubstituted 2-deoxystreptamine aminoglycoside antibiotics, comprising kanamycin A, kanamycin B, tobramycin, and dibekacin. These compounds interfere with protein synthesis by targeting the ribosomal decoding A site, and they differ in the numbers and locations of amino and hydroxy groups of the glucopyranosyl moiety (ring I). We synthesized kanamycin analogues characterized by subtle variations of the 2' and 6' substituents of ring I. The functional activities of the kanamycins and the synthesized analogues were investigated (i) in cell-free translation assays on wild-type and mutant bacterial ribosomes to study drug-target interaction, (ii) in MIC assays to assess antibacterial activity, and (iii) in rabbit reticulocyte translation assays to determine activity on eukaryotic ribosomes. Position 2' forms an intramolecular H bond with O5 of ring II, helping the relative orientations of the two rings with respect to each other. This bond becomes critical for drug activity when a 6'-OH substituent is present.
Assuntos
Antibacterianos/farmacologia , Canamicina/análogos & derivados , Canamicina/farmacologia , Aminas/química , Animais , Antibacterianos/química , Sequência de Carboidratos , Hidroxilação , Canamicina/química , Luciferases/química , Luciferases/genética , Testes de Sensibilidade Microbiana , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium smegmatis/genética , RNA Bacteriano/genética , RNA Ribossômico/genética , Coelhos , Reticulócitos/efeitos dos fármacos , Ribossomos/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Drug resistance in Mycobacterium tuberculosis is a global problem, with major consequences for treatment and public health systems. As the emergence and spread of drug-resistant tuberculosis epidemics is largely influenced by the impact of the resistance mechanism on bacterial fitness, we wished to investigate whether compensatory evolution occurs in drug-resistant clinical isolates of M. tuberculosis. By combining information from molecular epidemiology studies of drug-resistant clinical M. tuberculosis isolates with genetic reconstructions and measurements of aminoglycoside susceptibility and fitness in Mycobacterium smegmatis, we have reconstructed a plausible pathway for how aminoglycoside resistance develops in clinical isolates of M. tuberculosis. Thus, we show by reconstruction experiments that base changes in the highly conserved A-site of 16S rRNA that: (i) cause aminoglycoside resistance, (ii) confer a high fitness cost and (iii) destabilize a stem-loop structure, are associated with a particular compensatory point mutation that restores rRNA secondary structure and bacterial fitness, while maintaining to a large extent the drug-resistant phenotype. The same types of resistance and associated mutations can be found in M. tuberculosis in clinical isolates, suggesting that compensatory evolution contributes to the spread of drug-resistant tuberculosis disease.
Assuntos
Aminoglicosídeos/farmacologia , Antituberculosos/farmacologia , Farmacorresistência Bacteriana , Evolução Molecular , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , RNA Ribossômico 16S/genética , Análise Mutacional de DNA , Humanos , Mutagênese , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/crescimento & desenvolvimento , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Seleção Genética , Tuberculose/microbiologiaRESUMO
Capreomycin and the structurally similar compound viomycin are cyclic peptide antibiotics which are particularly active against Mycobacterium tuberculosis, including multidrug resistant strains. Both antibiotics bind across the ribosomal interface involving 23S rRNA helix 69 (H69) and 16S rRNA helix 44 (h44). The binding site of tuberactinomycins in h44 partially overlaps with that of aminoglycosides, and they share with these drugs the side effect of irreversible hearing loss. Here we studied the drug target interaction on ribosomes modified by site-directed mutagenesis. We identified rRNA residues in h44 as the main determinants of phylogenetic selectivity, predict compensatory evolution to impact future resistance development, and propose mechanisms involved in tuberactinomycin ototoxicity, which may enable the development of improved, less-toxic derivatives.
Assuntos
Antituberculosos/farmacologia , Capreomicina/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Ribossomos/efeitos dos fármacos , Viomicina/farmacologia , Aminoglicosídeos/farmacologia , Antituberculosos/metabolismo , Antituberculosos/toxicidade , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Capreomicina/metabolismo , Capreomicina/toxicidade , Farmacorresistência Bacteriana Múltipla/genética , Enviomicina/análogos & derivados , Enviomicina/farmacologia , Enviomicina/toxicidade , Mutagênese Sítio-Dirigida , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , RNA Ribossômico 16S/metabolismo , RNA Ribossômico 23S/metabolismo , Viomicina/metabolismo , Viomicina/toxicidadeRESUMO
Translation fidelity is the limiting factor in the accuracy of gene expression. With an estimated frequency of 10-4, errors in mRNA decoding occur in a mostly stochastic manner. Little is known about the response of higher eukaryotes to chronic loss of ribosomal accuracy as per an increase in the random error rate of mRNA decoding. Here, we present a global and comprehensive picture of the cellular changes in response to translational accuracy in mammalian ribosomes impaired by genetic manipulation. In addition to affecting established protein quality control pathways, such as elevated transcript levels for cytosolic chaperones, activation of the ubiquitin-proteasome system, and translational slowdown, ribosomal mistranslation led to unexpected responses. In particular, we observed increased mitochondrial biogenesis associated with import of misfolded proteins into the mitochondria and silencing of the unfolded protein response in the endoplasmic reticulum.
Assuntos
Biogênese de Organelas , Ribossomos/genética , Ribossomos/metabolismo , Resposta a Proteínas não Dobradas/genética , Substituição de Aminoácidos , Retículo Endoplasmático/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Mitocôndrias/metabolismo , Mutação , Biossíntese de Proteínas , Transporte Proteico/genética , Proteostase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismoRESUMO
The ribosomal protein S8 plays a pivotal role in the assembly of the 30S ribosomal subunit. Using filter binding assays, S8 proteins from mesophilic, and (hyper)thermophilic species of the archaeal genus Methanococcus and from the bacteria Escherichia coli and Thermus thermophilus were tested for their affinity to their specific 16S rRNA target site. S8 proteins from hyperthermophiles exhibit a 100-fold and S8 from thermophiles exhibit a 10-fold higher affinity than their mesophilic counterparts. Thus, there is a striking correlation of affinity of S8 proteins for their specific RNA binding site and the optimal growth temperatures of the respective organisms. The stability of individual rRNA-protein complexes might modulate the stability of the ribosome, providing a maximum of thermostability and flexibility at the growth temperature of the organism.
Assuntos
Archaea/crescimento & desenvolvimento , Bactérias/crescimento & desenvolvimento , RNA Ribossômico 16S/metabolismo , Proteínas Ribossômicas/metabolismo , Sequência de Aminoácidos , Archaea/química , Archaea/genética , Bactérias/química , Bactérias/genética , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Mathanococcus/química , Mathanococcus/genética , Mathanococcus/crescimento & desenvolvimento , Ligação Proteica , Estabilidade de RNA , RNA Ribossômico 16S/química , Alinhamento de Sequência , Temperatura , Thermus thermophilus/química , Thermus thermophilus/genética , Thermus thermophilus/crescimento & desenvolvimentoRESUMO
Clinical use of 2-deoxystreptamine aminoglycoside antibiotics, which target the bacterial ribosome, is compromised by adverse effects related to limited drug selectivity. Here we present a series of 4',6'-O-acetal and 4'-O-ether modifications on glucopyranosyl ring I of aminoglycosides. Chemical modifications were guided by measuring interactions between the compounds synthesized and ribosomes harbouring single point mutations in the drug-binding site, resulting in aminoglycosides that interact poorly with the drug-binding pocket of eukaryotic mitochondrial or cytosolic ribosomes. Yet, these compounds largely retain their inhibitory activity for bacterial ribosomes and show antibacterial activity. Our data indicate that 4'-O-substituted aminoglycosides possess increased selectivity towards bacterial ribosomes and little activity for any of the human drug-binding pockets.
Assuntos
Aminoglicosídeos/química , Aminoglicosídeos/farmacologia , Antibacterianos/farmacologia , Aminoglicosídeos/uso terapêutico , Animais , Antibacterianos/uso terapêutico , Sequência de Bases , Sistema Livre de Células , Cristalografia por Raios X , Modelos Animais de Doenças , Interações Medicamentosas , Escherichia coli/efeitos dos fármacos , Escherichia coli/isolamento & purificação , Humanos , Concentração Inibidora 50 , Masculino , Camundongos , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Mycobacterium smegmatis/efeitos dos fármacos , Conformação de Ácido Nucleico , Biossíntese de Proteínas/efeitos dos fármacos , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética , Ribossomos/metabolismo , Sepse/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacosRESUMO
Aminoglycoside antibiotics target the ribosomal decoding A-site and are active against a broad spectrum of bacteria. These compounds bind to a highly conserved stem-loop-stem structure in helix 44 of bacterial 16S rRNA. One particular aminoglycoside, paromomycin, also shows potent antiprotozoal activity and is used for the treatment of parasitic infections, e.g. by Leishmania spp. The precise drug target is, however, unclear; in particular whether aminoglycoside antibiotics target the cytosolic and/or the mitochondrial protozoan ribosome. To establish an experimental model for the study of protozoan decoding-site function, we constructed bacterial chimeric ribosomes where the central part of bacterial 16S rRNA helix 44 has been replaced by the corresponding Leishmania and Trypanosoma rRNA sequences. Relating the results from in-vitro ribosomal assays to that of in-vivo aminoglycoside activity against Trypanosoma brucei, as assessed in cell cultures and in a mouse model of infection, we conclude that aminoglycosides affect cytosolic translation while the mitochondrial ribosome of trypanosomes is not a target for aminoglycoside antibiotics.