Enhancing the therapeutic window for Spectinamide anti-tuberculosis Agents: Synthesis, Evaluation, and activation of phosphate prodrug 3408.

Spectinamides are a novel class of narrow-spectrum antitubercular agents with the potential to treat drug-resistant tuberculosis infections. Spectinamide 1810 has shown a good safety record following subcutaneous injection in mice or infusion in rats but exhibits transient acute toxicity following bolus administration in either species. To improve the therapeutic index of 1810, an injectable prodrug strategy was explored. The injectable phosphate prodrug 3408 has a superior maximum tolerated dose compared to 1810 or Gentamicin. Following intravenous administration in rodents, prodrug 3408 was quickly converted to 1810. The resulting 1810 exposure and pharmacokinetic profile after 3408 administration was identical to equivalent molar amounts of 1810 given directly by intravenous administration. 3408 and the parent 1810 exhibited similar overall efficacy in a BALB/c acute tuberculosis efficacy model. Delivery of 1810 in phosphate prodrug form, therefore, holds the potential to improve further the therapeutic index of an already promising tuberculosis antibiotic.

Mitochondrial misreading in skeletal muscle accelerates metabolic aging and confers lipid accumulation and increased inflammation.

We have recently reported on an experimental model of mitochondrial mistranslation conferred by amino acid exchange V338Y in the mitochondrial ribosomal protein MrpS5. Here we used a combination of RNA-Seq and metabolic profiling of homozygous transgenic MrpS5V338Y/V338Y mice to analyze the changes associated with the V338Y mutation in post-mitotic skeletal muscle. Metabolic profiling demonstrated age-dependent metabolic changes in the mutant V338Y animals, which included enhanced levels of age-associated metabolites and which were accompanied by increased glycolysis, lipid desaturation and eicosanoid biosynthesis, and alterations of the pentose phosphate pathway. In addition, transcriptome signatures of aged V338Y mutant muscle pointed to elevated inflammation, likely reflecting the increased levels of bioactive lipids. Our findings indicate that mistranslation-mediated chronic impairment of mitochondrial function affects specific bioenergetic processes in muscle in an age-dependent manner.

Aminoglycoside-modifying enzymes determine the innate susceptibility to aminoglycoside antibiotics in rapidly growing mycobacteria.

OBJECTIVES: Infections caused by the rapidly growing mycobacterium (RGM) Mycobacterium abscessus are notoriously difficult to treat due to the innate resistance of M. abscessus to most clinically available antimicrobials. Aminoglycoside antibiotics (AGA) are a cornerstone of antimicrobial chemotherapy against M. abscessus infections, although little is known about intrinsic drug resistance mechanisms. We investigated the role of chromosomally encoded putative aminoglycoside-modifying enzymes (AME) in AGA susceptibility in M. abscessus. METHODS: Clinical isolates of M. abscessus were tested for susceptibility to a series of AGA with different substituents at positions 2', 3' and 4' of ring 1 in MIC assays. Cell-free extracts of M. abscessus type strain ATCC 19977 and Mycobacterium smegmatis strains SZ380 [aac(2')-Id(+)], EP10 [aac(2')-Id(-)] and SZ461 [aac(2')-Id(+), rrs A1408G] were investigated for AGA acetylation activity using thin-layer chromatography (TLC). Cell-free ribosome translation assays were performed to directly study drug-target interaction. RESULTS: Cell-free translation assays demonstrated that ribosomes of M. abscessus and M. smegmatis show comparable susceptibility to all tested AGA. MIC assays for M. abscessus and M. smegmatis, however, consistently showed the lowest MIC values for 2'-hydroxy-AGA as compared with 2'-amino-AGA, indicating that an aminoglycoside-2'-acetyltransferase, Aac(2'), contributes to innate AGA susceptibility. TLC experiments confirmed enzymatic activity consistent with Aac(2'). Using M. smegmatis as a model for RGM, acetyltransferase activity was shown to be up-regulated in response to AGA-induced inhibition of protein synthesis. CONCLUSIONS: Our findings point to AME as important determinants of AGA susceptibility in M. abscessus.

Structure-Activity Relationships of Spectinamide Antituberculosis Agents: A Dissection of Ribosomal Inhibition and Native Efflux Avoidance Contributions.

Spectinamides are a novel class of antitubercular agents with the potential to treat drug-resistant tuberculosis infections. Their antitubercular activity is derived from both ribosomal affinity and their ability to overcome intrinsic efflux mediated by the Mycobacterium tuberculosis Rv1258c efflux pump. This study explores the structure-activity relationships through analysis of 50 targeted spectinamides. Compounds are evaluated for ribosomal translational inhibition, MIC activity in Rv1258c efflux pump deficient and wild type tuberculosis strains, and efficacy in an acute model of tuberculosis infection. The results of this study show a narrow structure-activity relationship, consistent with a tight ribosome-binding pocket and strict structural requirements to overcome native efflux. Rationalization of ribosomal inhibition data using molecular dynamics simulations showed stable complex formation for halogenated spectinamides consistent with the long post antibiotic effects observed. The lead spectinamides identified in this study demonstrated potent MIC activity against MDR and XDR tuberculosis and had desirable antitubercular class specific features including low protein binding, low microsomal metabolism, no cytotoxicity, and significant reductions in bacterial burdens in the lungs of mice infected with M. tuberculosis. The structure-activity relationships detailed here emphasize the need to examine efflux-mediated resistance in the design of antituberculosis drugs and demonstrate that it is possible to overcome intrinsic efflux with synthetic modification. The ability to understand the structure requirements for this class has produced a variety of new substituted spectinamides, which may provide useful alternative candidates and promote the further development of this class.

In vitro and in vivo Evaluation of Synergism between Anti-Tubercular Spectinamides and Non-Classical Tuberculosis Antibiotics.

Spectinamides are new semi-synthetic spectinomycin derivatives with potent anti-tubercular activity. The reported synergism of the precursor spectinomycin with other antibiotics prompted us to examine whether spectinamides sensitize M. tuberculosis to other antibiotics not traditionally used in the treatment of tuberculosis to potentially expand therapeutic options for MDR/XDR Tuberculosis. Whole cell synergy checkerboard screens were performed using the laboratory strain M. tuberculosis H37Rv, lead spectinamide 1599, and a broad panel of 27 antibiotics. In vitro, 1599 synergized with 11 drugs from 6 antibiotic classes. The observed synergy was tested against clinical isolates confirming synergy with Clarithromycin, Doxycycline and Clindamycin, combinations of which were taken forward for in vivo efficacy determination. Co-administration of 1599 and clarithromycin provided additional bacterial killing in a mouse model of acute tuberculosis infection, but not in a chronic infection model. Further studies indicated that mismatched drug exposure profiles likely permitted induction of phenotypic clarithromycin resistance and subsequent loss of synergism. These studies highlight the importance of validating in vitro synergism and the challenge of matching drug exposures to obtain a synergistic outcome in vivo. Results from this study indicate that a 1599 clarithromycin combination is potentially viable, providing the drug exposures can be carefully monitored.