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.antibiotic | translation | misreading | deafness | mycobacteria

Aminoglycoside ototoxicity has been related to a surprisingly large number of cellular structures and metabolic pathways. The finding that patients with mutations in mitochondrial rRNA are hypersusceptible to aminoglycoside-induced hearing loss has indicated a possible role for mitochondrial protein synthesis. To study the molecular interaction of aminoglycosides with eukaryotic ribosomes, we made use of the observation that the drug binding site is a distinct domain defined by the small subunit rRNA, and investigated drug susceptibility of bacterial hybrid ribosomes carrying various alleles of the eukaryotic decoding site. Compared to hybrid ribosomes with the A site of human cytosolic ribosomes, susceptibility of mitochondrial hybrid ribosomes to various aminoglycosides correlated with the relative cochleotoxicity of these drugs. Sequence alterations that correspond to the mitochondrial deafness mutations A1555G and C1494T increased drug-binding and rendered the ribosomal decoding site hypersusceptible to aminoglycoside-induced mistranslation and inhibition of protein synthesis. Our results provide experimental support for aminoglycoside-induced dysfunction of the mitochondrial ribosome. We propose a pathogenic mechanism in which interference of aminoglycosides with mitochondrial protein synthesis exacerbates the drugs' cochlear toxicity, playing a key role in sporadic dosedependent and genetically inherited, aminoglycoside-induced deafness.decoding ͉ mitochondria ͉ ribosomes ͉ toxicity ͉ translation

Despite the fact that important genetic diseases are caused by mutant mitochondrial ribosomes, the molecular mechanisms by which such ribosomes result in a clinical phenotype remain largely unknown. The absence of experimental models for mitochondrial diseases has also prevented the rational search for therapeutic interventions. Here, we report on the construction of bacterial hybrid ribosomes that contain various versions of the mitochondrial decoding region of ribosomal RNA. We show that the pathogenic mutations A1555G and C1494T decrease the accuracy of translation and render the ribosomal decoding site hypersusceptible to aminoglycoside antibiotics. This finding suggests misreading of the genetic code as an important molecular mechanism in disease pathogenesis.decoding ͉ mitochondria ͉ mutant rRNA ͉ ribosomes ͉ disease

SummaryDrug 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.

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.

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.

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.

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, lesstoxic derivatives.Tuberculosis has been declared a global public health emergency by the World Health Organization. In 2000, worldwide there were about 9 million new cases and 2 million deaths due to tuberculosis (10). The emergence of multidrug-resistant (MDR) tuberculosis has further complicated treatment and control of the disease (1). Capreomycin and viomycin belong to the tuberactinomycins, an important class of antibiotics with activities against multidrug-resistant tuberculosis (12).Tuberactinomycins are cyclic peptide antibiotics (for chemical structures, see Fig. S1 in the supplemental material) which target bacterial protein synthesis by binding to the well-conserved intersubunit bridge B2a, formed by interaction between helix 69 (H69) of the 23S rRNA and helix 44 (h44) of the 16S rRNA ( Fig. 1A to C) (34). Inhibition of translocation during peptide elongation is the main mechanism of drug action mediating the compound's antibacterial activity (24,26). Mutational alterations of nucleotides in 23S or 16S rRNA affect drug binding (8,13,18,20,37). In addition, the loss of 2Ј-Omethylation of C1920 (rRNA nucleotides are numbered according to those for Escherichia coli throughout the paper) in H69 and that of C1409 in h44 by TlyA reduces susceptibility to capreomycin (17, 21). Interestingly, Thermus thermophilus TlyA modifies only C1920 in H69 of 23S rRNA, but not C1409 in h44 of 16S rRNA. Inactivation of tlyA in T. thermophilus does not affect its sensitivity to capreomycin (25), suggesting that modification of C1409 is the relevant determinant of increased drug susceptibility due to 2Ј-O-methylation.Much of the unwanted side effects of ribosomal antibiotics reflects limitations in selectivity (2, 3), as has been demonstrated for chloramphenicol and linezolid (23,38). Together with the structurally unrelated class of aminoglycoside antibiotics, the tuberactinomycins share ototoxicity (irreversible loss of hearing) as a common and unique side effect (7, 35). Tuberactinomycins affect bacterial protein synthesis apparently by stabilizing peptidyl-tRNA in the pretranslocation complex, preventing translocation of the ribosome along the mRNA (11,19,24,26). The recent crystal...