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.

Spectinamides: a new class of semisynthetic antituberculosis agents that overcome native drug efflux.

Although the classical antibiotic spectinomycin is a potent bacterial protein synthesis inhibitor, poor antimycobacterial activity limits its clinical application for treating tuberculosis. Using structure-based design, we generated a new semisynthetic series of spectinomycin analogs with selective ribosomal inhibition and excellent narrow-spectrum antitubercular activity. In multiple murine infection models, these spectinamides were well tolerated, significantly reduced lung mycobacterial burden and increased survival. In vitro studies demonstrated a lack of cross resistance with existing tuberculosis therapeutics, activity against multidrug-resistant (MDR) and extensively drug-resistant tuberculosis and an excellent pharmacological profile. Key to their potent antitubercular properties was their structural modification to evade the Rv1258c efflux pump, which is upregulated in MDR strains and is implicated in macrophage-induced drug tolerance. The antitubercular efficacy of spectinamides demonstrates that synthetic modifications to classical antibiotics can overcome the challenge of intrinsic efflux pump-mediated resistance and expands opportunities for target-based tuberculosis drug discovery.

The integrated response of primary metabolites to gene deletions and the environment.

Intracellular metabolites arise from the molecular integration of genomic and environmental factors that jointly determine metabolic activity. However, it is not clear how the interplay of genotype, nutrients, growth, and fluxes affect metabolite concentrations globally. Here we used quantitative metabolomics to assess the combined effect of environment and genotype on the metabolite composition of a yeast cell. We analyzed a panel of 34 yeast single-enzyme knockout mutants grown on three archetypical carbon sources, generating a dataset of 400 unique metabolome samples. The different carbon sources globally affected the concentrations of intermediates, both directly, by changing the thermodynamic potentials (Δ(r)G) as a result of the substrate influx, and indirectly, by cellular regulation. In contrast, enzyme deletion elicited only local accumulation of the metabolic substrate immediately upstream of the lesion. Key biosynthetic precursors and cofactors were generally robust under all tested perturbations in spite of changes in fluxes and growth rate.

Structure-activity relationships among the kanamycin aminoglycosides: role of ring I hydroxyl and amino groups.

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.

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.

Molecular basis for the selectivity of antituberculosis compounds capreomycin and viomycin.

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.

Directed mutagenesis of Mycobacterium smegmatis 16S rRNA to reconstruct the in vivo evolution of aminoglycoside resistance in Mycobacterium tuberculosis.

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.

SUMO-independent in vivo activity of a SUMO-targeted ubiquitin ligase toward a short-lived transcription factor.

Many proteins are regulated by ubiquitin-dependent proteolysis. Substrate ubiquitylation can be stimulated by additional post-translational modifications, including small ubiquitin-like modifier (SUMO) conjugation. The recently discovered SUMO-targeted ubiquitin ligases (STUbLs) mediate the latter effect; however, no endogenous substrates of STUbLs that are degraded under normal conditions are known. From a targeted genomic screen, we now identify the yeast STUbL Slx5-Slx8, a heterodimeric RING protein complex, as a key ligase mediating degradation of the MATalpha2 (alpha2) repressor. The ubiquitin-conjugating enzyme Ubc4 was found in the same screen. Surprisingly, mutants with severe defects in SUMO-protein conjugation were not impaired for alpha2 turnover. Unmodified alpha2 also bound to and was ubiquitylated efficiently by Slx5-Slx8. Nevertheless, when we inactivated four SUMO-interacting motifs (SIMs) in Slx5 that together account for its noncovalent SUMO binding, both in vitro Slx5-Slx8-dependent ubiquitylation and in vivo degradation of alpha2 were inhibited. These data identify alpha2 as the first native substrate of the conserved STUbLs, and demonstrate that its STUbL-mediated ubiquitylation does not require SUMO. We suggest that alpha2, and presumably other proteins, have surface features that mimic SUMO, and therefore can directly recruit STUbLs without prior SUMO conjugation.