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, matrix-associated, 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.

Keywords: cancer; gentamicin B1; nonsense mutation; premature stop codon readthrough; rare genetic diseases.

Fig. 1.

PTC readthrough by gentamicin components. (A, B, D, and F) The proportion of HDQ-P1 cells showing nuclear p53 immunofluorescence, a measure of readthrough, after a 72-h exposure to different concentrations of three gentamicin batches or the indicated compounds was determined by automated fluorescence microscopy (7). A and F show representative images, with p53 immunofluorescence in green and nuclei in blue. (Scale bar, 100 µm.) Complete sets of images are shown in Fig. S1. B and D show quantitative measurements in ∼2,000 cells per replicate (mean ± SD, n = 3). *P < 0.01 relative to untreated samples. (C and E) Structures of the major gentamicins and of gentamicin B1 and B ( in C, ^§C2 and C2a are epimers at the ring I 6′ position). (G) The production of full-length p53 (FL-p53) and truncated p53 (TR-p53) in HDQ-P1 cells exposed for 72 h to different concentrations of three gentamicin batches or B1 was determined by automated capillary electrophoresis Western analysis and the results are displayed as pseudoblots (7). Vinculin was used as a protein loading control. The numbers indicate the amounts of normalized FL-p53 against vinculin relative to the amount of TR-p53 detected in untreated cells. (H) R213X TP53 mRNA was subjected to in vitro translation in the presence of the indicated compounds at 0.5 µM for 20 min and p53 was detected as in G.

Fig. S1.

PTC readthrough by gentamicin components. The images show the effect of exposure of HDQ-P1 cells for 72 h to different concentrations of three gentamicin batches (A), gentamicin B1 (B), or the indicated gentamicin components (C) on p53 levels, determined by automated immunofluorescence microscopy. p53 immunofluorescence is shown in green and nuclei in blue. (Scale bar, 100 µm.)

Fig. 2.

Conformations of A1824 and A1825 upon binding of G418, gentamicin B1, and gentamicin B. (A–C) Overview of the effect of binding of G418 (A, green, pdb: 4U4O), gentamicin B1 (B, purple, MD simulation), and gentamicin B (C, blue, MD simulation) to rRNA helix 44. B and C depict the most important conformations during the 1-ns MD simulation. The rRNA is shown as cartoon except for A1824 and A1825, which are shown as sticks. A1824 and A1825 are flipped out, in an “elongation-like” conformation in the presence of G418 or B1, whereas only A1825 is flipped out, in a “termination-like” conformation, in the presence of gentamicin B. (D–F) Close-up view of the areas boxed in A–C (rotated ∼90° around the y axis), illustrating interactions (dashed lines) between ring I and rRNA. (G) Distance between atom N₆ of A1824 and atom O₂ of C1710 on the opposite side of the helix during the course of the simulation (Fig. S2 for conformations in control simulations without and with G418).

Fig. S2.

MD control simulations of the conformation of A1824 and A1825 with and without G418. Depicted are the most important conformations of rRNA helix 44 bound to G418 (A, green) and without a compound (B) during the course of the MD simulation. The rRNA is shown as cartoon except for A1824 and A1825, which are shown as sticks. A1824 and A1825 are flipped out, in an “elongation-like” conformation in the presence of G418 (A) as observed in the crystal structure (pdb: 4U4O) but both move in when no compound is bound, as indicated by the curved arrows (B).

Fig. 3.

PTC readthrough in cell culture and in vivo. (A) PTC readthrough in TP53-null NCI-H1299 cells transiently transfected with TP53 R213X TGA, TAG, or TAA constructs and exposed for 48 h to B1 or gentamicin (Gent.). Full-length and truncated p53 were quantified relative to the amount of p53 in untreated cells. p53-WT: R213; mock: transfection reagents only. (B) PTC readthrough in cancer cell lines with different TP53 nonsense mutations exposed to gentamicin B1. The mutations are shown in parentheses. Vinculin was used as a protein loading control. Red arrowhead: full-length p53; black arrowhead: truncated p53. (C) PTC readthrough in NRG mice bearing xenografts of NCI-H1299 cells stably expressing TP53 R213X . Each lane is from an individual mouse xenograft. (Left) Mice were injected once with saline or the indicated concentrations of B1 or USP gentamicin and the amounts of truncated p53 and full-length p53 were measured 48 h later. *These mice were killed shortly after administration because of severe toxicity. (Right) Mice were injected with the indicated doses on 5 consecutive days and p53 levels were measured 72 h after the last injection. Vinculin was used as a protein loading control. The numbers below the panels indicate the amounts of normalized FL-p53 against vinculin relative to the ones of saline-treated samples.

Fig. S3.

Induction of PTC readthrough by gentamicin B1 in NCI-H1299 cells stably expressing TP53 R213X. The production of full-length p53 (FL-p53) and truncated p53 (TR-p53) after 72 h exposure to the indicated concentrations of gentamicin B1 was determined by automated capillary electrophoresis Western analysis and the results are displayed as pseudoblots. The numbers indicate the amounts of FL-p53 relative to the amount of FL-p53 detected in untreated cells.

Fig. 4.

Induction of PTC readthrough by gentamicin B1 in cells derived from patients with rare genetic diseases. (A and B) TPP1 enzyme activity and protein levels measured after exposure of GM16485 fibroblasts with TPP1 nonsense mutations to B1 or gentamicin for up to 10 d. (A) TPP1 activity was determined and expressed relative to the average activity of untreated primary fibroblasts from two unaffected individuals (WT). (B) The same cell extracts were analyzed for production of TPP1 by automated capillary electrophoresis Western analysis. Extracts from WT fibroblasts were also analyzed, using 20% of the amount of protein used for GM16485. Vinculin was used as a loading control. (C) Full-length dystrophin protein levels measured after HSK001 myoblasts with a DMD nonsense mutation were differentiated into myotubes and exposed to B1 or gentamicin for 3 d. Extracts from WT myotubes were also analyzed, using 5% of the amount of protein used for Duchenne muscular dystrophy (DMD) cells. β-Actin was used as a loading control. (D) Full-length SMARCAL1 protein levels measured after exposure of SD123 fibroblasts with SMARCAL1 nonsense mutations to gentamicin B1 or gentamicin for 6 d. Extracts from WT fibroblasts were also analyzed, using 10% of the amount of protein used for SIOD cells. β-Actin was used as a loading control. (E) Full-length collagen VII measured after exposure of EB14 keratinocytes with COL7A1 nonsense mutations to B1 or gentamicin for 72 h. Extracts from WT keratinocytes were also analyzed, using 20% of the amount of protein used for EB14 cells. β-Actin was used as a loading control.

Fig. S4.

Effect of gentamicin B1 on cell viability. HDQ-P1 (A), NCI-H1299 (B), immortalized human fibroblast (C), C25CI48 (myoblast) (D), and K1 (keratinocyte) (E) cells were exposed to gentamicin B1 or gentamicin sulfate for up to 72 h in triplicate and cell viability was determined using the MTT assay.

Fig. S5.

PTC readthrough by gentamicin B1 is reduced by other gentamicin components. HDQ-P1 cells were exposed in duplicate to the indicated concentrations of gentamicin sulfate (A and B) or purified individual gentamicin components (C and D) without or with gentamicin B1 for 72 h. The percentage of p53⁺ nuclei was determined by automated immunofluorescence microscopy (A and C) and cell viability was determined using the MTT assay (B and D).