David M. Bedwell

Aminoglycoside antibiotics mediate context-dependent suppression of termination codons in a mammalian translation system.

The translation machinery recognizes codons that enter the ribosomal A site with remarkable accuracy to ensure that polypeptide synthesis proceeds with a minimum of errors. When a termination codon enters the A site of a eukaryotic ribosome, it is recognized by the release factor eRF1. It has been suggested that the recognition of translation termination signals in these organisms is not limited to a simple trinucleotide codon, but is instead recognized by an extended tetranucleotide termination signal comprised of the stop codon and the first nucleotide that follows. Interestingly, pharmacological agents such as aminoglycoside antibiotics can reduce the efficiency of translation termination by a mechanism that alters this ribosomal proofreading process. This leads to the misincorporation of an amino acid through the pairing of a near-cognate aminoacyl tRNA with the stop codon. To determine whether the sequence context surrounding a stop codon can influence aminoglycoside-mediated suppression of translation termination signals, we developed a series of readthrough constructs that contained different tetranucleotide termination signals, as well as differences in the three bases upstream and downstream of the stop codon. Our results demonstrate that the sequences surrounding a stop codon can play an important role in determining its susceptibility to suppression by aminoglycosides. Furthermore, these distal sequences were found to influence the level of suppression in remarkably distinct ways. These results suggest that the mRNA context influences the suppression of stop codons in response to subtle differences in the conformation of the ribosomal decoding site that result from aminoglycoside binding.

PTC124 is an orally bioavailable compound that promotes suppression of the human CFTR-G542X nonsense allele in a CF mouse model

Nonsense mutations inactivate gene function and are the underlying cause of a large percentage of the individual cases of many genetic disorders. PTC124 is an orally bioavailable compound that promotes readthrough of premature translation termination codons, suggesting that it may have the potential to treat genetic diseases caused by nonsense mutations. Using a mouse model for cystic fibrosis (CF), we show that s.c. injection or oral administration of PTC124 to Cftr−/− mice expressing a human CFTR-G542X transgene suppressed the G542X nonsense mutation and restored a significant amount of human (h)CFTR protein and function. Translational readthrough of the premature stop codon was demonstrated in this mouse model in two ways. First, immunofluorescence staining showed that PTC124 treatment resulted in the appearance of hCFTR protein at the apical surface of intestinal glands in Cftr−/− hCFTR-G542X mice. In addition, functional assays demonstrated that PTC124 treatment restored 24–29% of the average cAMP-stimulated transepithelial chloride currents observed in wild-type mice. These results indicate that PTC124 can effectively suppress the hCFTR-G542X nonsense mutation in vivo. In light of its oral bioavailability, safety toxicology profile in animal studies, and efficacy with other nonsense alleles, PTC124 has the potential to be an important therapeutic agent for the treatment of inherited diseases caused by nonsense mutations.

GTP Hydrolysis by eRF3 Facilitates Stop Codon Decoding during Eukaryotic Translation Termination

ABSTRACT Translation termination in eukaryotes is mediated by two release factors, eRF1 and eRF3. eRF1 recognizes each of the three stop codons (UAG, UAA, and UGA) and facilitates release of the nascent polypeptide chain. eRF3 is a GTPase that stimulates the translation termination process by a poorly characterized mechanism. In this study, we examined the functional importance of GTP hydrolysis by eRF3 in Saccharomyces cerevisiae. We found that mutations that reduced the rate of GTP hydrolysis also reduced the efficiency of translation termination at some termination signals but not others. As much as a 17-fold decrease in the termination efficiency was observed at some tetranucleotide termination signals (characterized by the stop codon and the first following nucleotide), while no effect was observed at other termination signals. To determine whether this stop signal-dependent decrease in the efficiency of translation termination was due to a defect in either eRF1 or eRF3 recycling, we reduced the level of eRF1 or eRF3 in cells by expressing them individually from the CUP1 promoter. We found that the limitation of either factor resulted in a general decrease in the efficiency of translation termination rather than a decrease at a subset of termination signals as observed with the eRF3 GTPase mutants. We also found that overproduction of eRF1 was unable to increase the efficiency of translation termination at any termination signals. Together, these results suggest that the GTPase activity of eRF3 is required to couple the recognition of translation termination signals by eRF1 to efficient polypeptide chain release.

Aminoglycoside suppression of a premature stop mutation in a Cftr–/– mouse carrying a human CFTR-G542X transgene

Abstract. Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Since ~5% of all mutant CF alleles are stop mutations, it can be calculated that ~10% of CF patients carry a premature stop mutation in at least one copy of the CFTR gene. Certain ethnic groups, such as the Ashkenazi Jewish population, carry a much higher percentage of CF stop mutations. Consequently, a therapeutic strategy aimed at suppressing this class of mutation would be highly desirable for the treatment of this common genetic disease. We have shown previously that aminoglycoside antibiotics can suppress premature stop mutations in the CFTR gene in a bronchial epithelial cell line [Nat Med (1997) 3:1280]. To address whether aminoglycosides can suppress a CFTR premature stop mutation in an animal model, we constructed a transgenic mouse with a null mutation in the endogenous CFTR locus (Cftr–/–) that also expressed a human CFTR-G542X cDNA under control of the intestinal fatty acid binding protein promoter. We then investigated whether the daily administration of the aminoglycoside antibiotics gentamicin or tobramycin could restore the expression of a detectable level of CFTR protein. Immunofluorescence staining of intestinal tissues from Cftr–/–hCFTR-G542X mice revealed that gentamicin treatment resulted in the appearance of hCFTR protein at the apical surface of the glands of treated mice. Weaker staining was also observed in the intestinal glands following tobramycin treatment. Short-circuit current measurements made on intestinal tissues from these mice demonstrated that a significant number of positive cAMP-stimulated transepithelial chloride current measurements could be observed following gentamicin treatment (P=0.008) and a near significant number following tobramycin treatment (P=0.052). When taken together, these results indicate that gentamicin, and to a lesser extent tobramycin, can restore the synthesis of functional hCFTR protein by suppressing the hCFTR-G542X premature stop mutation in vivo.

The vacuolar Ca2+/H+ exchanger Vcx1p/Hum1p tightly controls cytosolic Ca2+ levels in S. cerevisiae.

It is well established that the vacuole plays an important role in the cellular adaptation to growth in the presence of elevated extracellular Ca2+ concentrations in Saccharomyces cerevisiae. The Ca2+ ATPase Pmc1p and the Ca2+/H+ exchanger Vcx1p/Hum1p have been shown to facilitate Ca2+ sequestration into the vacuole. However, the distinct physiological roles of these two vacuolar Ca2+ transporters remain uncertain. Here we show that Vcx1p can rapidly sequester a sudden pulse of cytosolic Ca2+ into the vacuole, while Pmc1p carries out this function much less efficiently. This finding is consistent with the postulated role of Vcx1p as a high capacity, low affinity Ca2+ transporter and suggests that Vcx1p may act to attenuate the propagation of Ca2+ signals in this organism.

Clinically relevant aminoglycosides can suppress disease-associated premature stop mutations in the IDUA and P53 cDNAs in a mammalian translation system.

Recent studies have suggested that the use of aminoglycosides to suppress disease-causing nonsense mutations may be a promising new therapy for a large number of genetic diseases. However, gentamicin is currently the only clinically relevant aminoglycoside shown to suppress premature stop mutations in a mammalian system. We compared the ability of the clinically approved aminoglycosides gentamicin, tobramycin, and amikacin to suppress premature stop mutations. Using readthrough reporter constructs as well as mammalian cDNAs containing naturally occurring premature stop mutations, we found that each of these aminoglycosides can suppress many premature stop mutations in a context-dependent manner in a mammalian translation system. Our results indicate that the tetranucleotide termination signal (the stop codon and the nucleotide 3′ of the stop codon) is the primary determinant for aminoglycoside-mediated suppression. The levels of termination suppression achieved by tobramycin were substantially lower than those observed with gentamicin. In contrast, amikacin stimulated suppression in a manner that was generally similar to gentamicin. Amikacin produced higher levels of readthrough than gentamicin at some contexts, demonstrating a unique pattern of context dependence. Experiments with mammalian cDNAs confirmed these results and demonstrated that these aminoglycosides can also suppress disease-associated premature stop mutations previously identified in the IDUA gene (responsible for the lysosomal storage disease mucopolysaccharidosis I) and the P53 gene (associated with many forms of cancer). Taken together, these results suggest that amikacin represents an alternative to gentamicin for suppression therapy in certain contexts, thus providing a means of optimizing the efficacy of aminoglycoside-mediated suppression of premature stop mutations.

Therapeutics Based on Stop Codon Readthrough

Nonsense suppression therapy encompasses approaches aimed at suppressing translation termination at in-frame premature termination codons (PTCs, also known as nonsense mutations) to restore deficient protein function. In this review, we examine the current status of PTC suppression as a therapy for genetic diseases caused by nonsense mutations. We discuss what is currently known about the mechanism of PTC suppression as well as therapeutic approaches under development to suppress PTCs. The approaches considered include readthrough drugs, suppressor tRNAs, PTC pseudouridylation, and inhibition of nonsense-mediated mRNA decay. We also discuss the barriers that currently limit the clinical application of nonsense suppression therapy and suggest how some of these difficulties may be overcome. Finally, we consider how PTC suppression may play a role in the clinical treatment of genetic diseases caused by nonsense mutations.

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

New drugs are needed to enhance premature termination codon (PTC) suppression to treat the underlying cause of cystic fibrosis (CF) and other diseases caused by nonsense mutations. We tested new synthetic aminoglycoside derivatives expressly developed for PTC suppression in a series of complementary CF models. Using a dual-luciferase reporter system containing the four most prevalent CF transmembrane conductance regulator (CFTR) nonsense mutations (G542X, R553X, R1162X, and W1282X) within their local sequence contexts (the three codons on either side of the PTC), we found that NB124 promoted the most readthrough of G542X, R1162X, and W1282X PTCs. NB124 also restored full-length CFTR expression and chloride transport in Fischer rat thyroid cells stably transduced with a CFTR-G542XcDNA transgene, and was superior to gentamicin and other aminoglycosides tested. NB124 restored CFTR function to roughly 7% of wild-type activity in primary human bronchial epithelial (HBE) CF cells (G542X/delF508), a highly relevant preclinical model with endogenous CFTR expression. Efficacy was further enhanced by addition of the CFTR potentiator, ivacaftor (VX-770), to airway cells expressing CFTR PTCs. NB124 treatment rescued CFTR function in a CF mouse model expressing a human CFTR-G542X transgene; efficacy was superior to gentamicin and exhibited favorable pharmacokinetic properties, suggesting that in vitro results translated to clinical benefit in vivo. NB124 was also less cytotoxic than gentamicin in a tissue-based model for ototoxicity. These results provide evidence that NB124 and other synthetic aminoglycosides provide a 10-fold improvement in therapeutic index over gentamicin and other first-generation aminoglycosides, providing a promising treatment for a wide array of CFTR nonsense mutations.

Clinical doses of amikacin provide more effective suppression of the human CFTR-G542X stop mutation than gentamicin in a transgenic CF mouse model

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause the disease cystic fibrosis. We previously reported that gentamicin administration suppressed a CFTR premature stop mutation in a Cftr−/− mouse model carrying a human CFTR-G542X (hCFTR-G542X) transgene, resulting in the appearance of hCFTR protein and function. However, the high doses used in that study resulted in peak serum levels well beyond the levels typically administered to humans. To address this problem, we identified doses of both gentamicin and amikacin that resulted in peak serum levels within their accepted clinical ranges. We then asked whether these doses could suppress the hCFTR-G542X mutation in the Cftr−/− hCFTR-G542X mouse model. Our results indicate that low doses of each compound restored some hCFTR protein expression and function, as shown by immunofluorescence and short-circuit current measurements. However, we found that amikacin suppressed the hCFTR-G542X premature stop mutation more effectively than gentamicin when administered at these clinically relevant doses. Because amikacin is also less toxic than gentamicin, it may represent a superior choice for suppression therapy in patients that carry a premature stop mutation in the CFTR gene.

Distinct eRF3 requirements suggest alternate eRF1 conformations mediate peptide release during eukaryotic translation termination.

Organisms that use the standard genetic code recognize UAA, UAG, and UGA as stop codons, whereas variant code species frequently alter this pattern of stop codon recognition. We previously demonstrated that a hybrid eRF1 carrying the Euplotes octocarinatus domain 1 fused to Saccharomyces cerevisiae domains 2 and 3 (Eo/Sc eRF1) recognized UAA and UAG, but not UGA, as stop codons. In the current study, we identified mutations in Eo/Sc eRF1 that restore UGA recognition and define distinct roles for the TASNIKS and YxCxxxF motifs in eRF1 function. Mutations in or near the YxCxxxF motif support the cavity model for stop codon recognition by eRF1. Mutations in the TASNIKS motif eliminated the eRF3 requirement for peptide release at UAA and UAG codons, but not UGA codons. These results suggest that the TASNIKS motif and eRF3 function together to trigger eRF1 conformational changes that couple stop codon recognition and peptide release during eukaryotic translation termination.