Increased Selectivity toward Cytoplasmic versus Mitochondrial Ribosome Confers Improved Efficiency of Synthetic Aminoglycosides in Fixing Damaged Genes: A Strategy for Treatment of Genetic Diseases Caused by Nonsense Mutations

Abstract

Compelling evidence is now available that gentamicin and geneticin (G418) can induce mammalian ribosome to suppress disease-causing nonsense mutations and partially restore the expression of functional proteins. However, toxicity and relative lack of efficacy at subtoxic doses limit the use of gentamicin for suppression therapy. Although G418 exhibits strongest activity, it is very cytotoxic even at low doses. We describe here the first systematic development of the novel aminoglycoside (S)-11 exhibiting similar in vitro and ex vivo activity to that of G418, while its cell toxicity is significantly lower than those of gentamicin and G418. Using a series of biochemical assays, we provide proof of principle that antibacterial activity and toxicity of aminoglycosides can be dissected from their suppression activity. The data further indicate that the increased specificity towards cytoplasmic ribosome correlates with the increased activity, and that the decreased specificity towards mitochondrial ribosome confers to the lowered cytotoxicity.

Identification of the molecular attributes required for aminoglycoside activity against Leishmania

“…Although no such potency gap for the two derivatives has been documented in wild-type bacterial strains, similar tendency has been reported in A1408G mutant bacteria (23), sharing greater binding site similarity with Leishmania. These results also correlate well with the higher miscoding (38) and premature termination codon suppressing potency reported for G418 over Paromomycin in eukaryotes (39).…”

Synthetic Aminoglycosides Efficiently Suppress Cystic Fibrosis Transmembrane Conductance Regulator Nonsense Mutations and Are Enhanced by Ivacaftor

“…However, a significant portion of the toxicity associated with aminoglycosides may stem from their ability to also inhibit mitochondrial translation. More recently, further advances in the rational design of these compounds has allowed their toxic effects to be separated from their ability to promote translational readthrough (22). In the current study, new synthetic aminoglycosides specifically developed to further enhance readthrough efficacy while maintaining their improved toxicity profile were evaluated in a series of cell-based and animal models for efficacy, toxicity, and pharmacokinetics ( Figure 1).…”

Design of Novel Aminoglycoside Derivatives with Enhanced Suppression of Diseases-Causing Nonsense Mutations

“…5 We have recently demonstrated that the observed significantly decreased prokaryotic ribosome specificity of 2 6 and 3 18 is also linked to their reduced mitochondrial translation inhibition and subsequently to their reduced ototoxic effects than gentamicin and G418. The observed similar impact on bacterial ribosome by the current (6 and 7) and previous leads (2 and 3) therefore suggest that 6 and 7 are perhaps less ototoxic than gentamicin and G418.…”

“…5 To test the validity of this trend, we examined comparative protein translation inhibition of pseudotrisaccharides 2, 3 and 6, 7 in eukaryotic system, using coupled transcription/translation Comparative in vitro stop codon suppression levels induced by compounds 2 (-△-), 6 (-▲-), and gentamicin (--■ --) (left) and by 3 (-Δ-), 7 (-▲-), and gentamicin (--■ --) (right) in a series of nonsense constructs representing various genetic diseases (shown in parentheses): (A) R3X (USH1), (B) R245X (USH1), (C) Q70X (HS), and (D) G542X (CF). The assays were performed as previously described by us.…”

“…Thus, even though instead of 6′-diastereomeric mixture reported in the latter study, we used the Pro ) were quantified in coupled transcription/translation assays by using active luciferase detection as previously described by us. 5 c Minimal inhibitory concentration (MIC) values were determined by using the double-microdilution method. pure 6′-diastereomers 1, 4, and 5, the data in Table 1 shows that 4 and 5 are 1.8-fold and 15.3-fold poorer inhibitors of prokaryotic translation than the parent compound 1 (IC 50 Pro values of 6.0, 11.0, and 91.9 μM for 1, 4, and 5, respectively).…”