Stefan Duscha

Dissociation of antibacterial activity and aminoglycoside ototoxicity in the 4-monosubstituted 2-deoxystreptamine apramycin.

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.

4′-O-substitutions determine selectivity of aminoglycoside antibiotics

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.

XBP1 mitigates aminoglycoside-induced endoplasmic reticulum stress and neuronal cell death

Here we study links between aminoglycoside-induced mistranslation, protein misfolding and neuropathy. We demonstrate that aminoglycosides induce misreading in mammalian cells and assess endoplasmic reticulum (ER) stress and unfolded protein response (UPR) pathways. Genome-wide transcriptome and proteome analyses revealed upregulation of genes related to protein folding and degradation. Quantitative PCR confirmed induction of UPR markers including C/EBP homologous protein, glucose-regulated protein 94, binding immunoglobulin protein and X-box binding protein-1 (XBP1) mRNA splicing, which is crucial for UPR activation. We studied the effect of a compromised UPR on aminoglycoside ototoxicity in haploinsufficient XBP1 (XBP1+/−) mice. Intra-tympanic aminoglycoside treatment caused high-frequency hearing loss in XBP1+/− mice but not in wild-type littermates. Densities of spiral ganglion cells and synaptic ribbons were decreased in gentamicin-treated XBP1+/− mice, while sensory cells were preserved. Co-injection of the chemical chaperone tauroursodeoxycholic acid attenuated hearing loss. These results suggest that aminoglycoside-induced ER stress and cell death in spiral ganglion neurons is mitigated by XBP1, masking aminoglycoside neurotoxicity at the organismal level.

Structure-Activity Relationships among the Kanamycin Aminoglycosides: Role of Ring I Hydroxyl and Amino Groups

ABSTRACT 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.

Molecular Basis for the Selectivity of Antituberculosis Compounds Capreomycin and Viomycin

ABSTRACT 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.

Identification and evaluation of improved 4'-O-(alkyl) 4,5-disubstituted 2-deoxystreptamines as next-generation aminoglycoside antibiotics.

ABSTRACT 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. 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.

Importance of the 6'-hydroxy group and its configuration for apramycin activity.

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.

Aminomethyl spectinomycins as therapeutics for drug-resistant respiratory tract and sexually transmitted bacterial infections.

A new series of spectinomycin analogs with potency against drug-resistant bacterial pathogens was designed and developed with a structure-based approach and validated in vitro and in vivo. Teaching an old antibiotic new tricks More and more cases of gonorrhea no longer respond to standard antibiotic treatment, leading the CDC (U.S. Centers for Disease Control and Prevention) to classify Neisseria gonorrhoeae as an urgent threat. New antibiotics are urgently needed to treat this and other emerging drug-resistant pathogens. To this end, Bruhn and Waidyarachchi et al. have taken a second look at an old group of antibiotics, the spectinomycins, a class of drugs that inhibit bacterial protein synthesis but do not kill many types of pathogens. By carefully mapping how the drug binds to the ribosome structure, the authors determined that N-benzyl–substituted spectinomycins should be able to inhibit the ribosomes of a broad spectrum of bacteria that produce disease. And indeed, this new series potently inhibited bacteria that cause respiratory illness (Streptococcus pneumoniae, Haemophilus influenzae, Legionella pneumophila, and Moraxella catarrhalis) and sexually transmitted disease (N. gonorrhoeae and Chlamydia trachomatis). Their pharmacokinetics properties were promising, and assays showed that they are unlikely to cause adverse reactions. These new spectinomycins are active against drug-resistant forms of S. pneumoniae and cure mice of fatal pneumococcal pneumonia and sepsis, an encouraging result for the eventual use of these drugs for human infection. 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.

Decoding and deafness: Two sides of a coin

Antibiotics used in clinical medicine for the treatment of infectious diseases frequently target bacterial protein synthesis, as illustrated by macrolides, ketolides, lincosamides, oxazolidinones, aminoglycosides, and tetracyclines (Gale et al., 1981). In general, antibiotics target the ribosome at sites of functional relevance, e. g. the sites of decoding, translocation, and peptidyl transfer. The emergence of antibiotic resistance and the toxicity associated with some of the available agents ask for a further exploitation of the ribosome as a drug target.

Mutant MRPS5 affects mitoribosomal accuracy and confers stress‐related behavioral alterations

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.