RETRACTED: Gentamicin B1 is a minor gentamicin component with major nonsense mutation suppression activity

Abstract

Nonsense mutations underlie about 10% of rare genetic disease cases. They introduce a premature termination codon (PTC) and prevent the formation of full-length protein. Pharmaceutical gentamicin, a mixture of several related aminoglycosides, is a frequently used antibiotic in humans that can induce PTC readthrough and suppress nonsense mutations at high concentrations. However, testing of gentamicin in clinical trials has shown that safe doses of this drug produce weak and variable readthrough activity that is insufficient for use as therapy. In this study we show that the major components of pharmaceutical gentamicin lack PTC readthrough activity but the minor component gentamicin B1 (B1) is a potent readthrough inducer. Molecular dynamics simulations reveal the importance of ring I of B1 in establishing a ribosome configuration that permits pairing of a near-cognate complex at a PTC. B1 induced readthrough at all three nonsense codons in cultured cancer cells with TP53 (tumor protein p53) mutations, in cells from patients with nonsense mutations in the TPP1 (tripeptidyl peptidase 1), DMD (dystrophin), SMARCAL1 (SWI/SNF-related, matrixassociated, actin-dependent regulator of chromatin, subfamily a-like 1), and COL7A1 (collagen type VII alpha 1 chain) genes, and in an in vivo tumor xenograft model. The B1 content of pharmaceutical gentamicin is highly variable and major gentamicins suppress the PTC readthrough activity of B1. Purified B1 provides a consistent and effective source of PTC readthrough activity to study the potential of nonsense suppression for treatment of rare genetic disorders.gentamicin B1 | nonsense mutation | premature stop codon readthrough | rare genetic diseases | cancer

Gentamicin B1 is not a minor gentamicin component with major nonsense mutation suppression activity

“…We previously reported that the major components of pharmaceutical gentamicin showed only weak PTC readthrough activity (8), corroborated recently by Friesen (9), but that the minor component B1was a potent readthrough compound (8). Here, we correct the record and show that the B1 used in our study was misidentified by the vendor and was in fact G418, and we provide evidence that synthetic gentamicin B1 is inactive as a readthrough compound.…”

“…We found that none of the major gentamicins showed significant activity, an observation recently corroborated by others (9). We reported that the minor component gentamicin B1 was 30-to 100fold more potent than the most potent gentamicin batches tested (8). In recent structure-activity relationship studies to be reported elsewhere, we observed that synthetic aminoglycoside analogs bearing ring 1 of gentamicin B1 lacked readthrough activity whereas analogs bearing ring 1 of the closely related aminoglycoside G418 showed readthrough activity.…”

“…In a 2017 publication, we examined this question by measuring the activity of pure samples of each individual major gentamicin component and of minor gentamicin components (8). We found that none of the major gentamicins showed significant activity, an observation recently corroborated by others (9).…”

“…The commercial gentamicin B1 samples used in our 2017 study (8), here termed B1-MCC, were purchased as free base and as sulfate salt. They were provided with Certificates of Analysis, mass spectrometry and NMR data ( Supplementary Figure 1) supporting designation as gentamicin B1, which was corroborated by an independent NMR analysis carried out at our request.…”

A new in vitro assay measuring direct interaction of nonsense suppressors with the eukaryotic protein synthesis machinery

“…EC 50 values found in intact cells differ considerably from those measured by our cosedimentation assay, being much higher for AGs, 23,30 and much lower for ataluren, 8,31 RTC13 25 and GJ072. 25 We attribute these differences to the poor uptake of positively charged aminoglycosides into cells, while uptake is favored for the hydrophobic ataluren-like molecules.…”