A small molecule that induces translational readthrough of CFTR nonsense mutations by eRF1 depletion.

Premature termination codons (PTCs) prevent translation of a full-length protein and trigger nonsense-mediated mRNA decay (NMD). Nonsense suppression (also termed readthrough) therapy restores protein function by selectively suppressing translation termination at PTCs. Poor efficacy of current readthrough agents prompted us to search for better compounds. An NMD-sensitive NanoLuc readthrough reporter was used to screen 771,345 compounds. Among the 180 compounds identified with readthrough activity, SRI-37240 and its more potent derivative SRI-41315, induce a prolonged pause at stop codons and suppress PTCs associated with cystic fibrosis in immortalized and primary human bronchial epithelial cells, restoring CFTR expression and function. SRI-41315 suppresses PTCs by reducing the abundance of the termination factor eRF1. SRI-41315 also potentiates aminoglycoside-mediated readthrough, leading to synergistic increases in CFTR activity. Combining readthrough agents that target distinct components of the translation machinery is a promising treatment strategy for diseases caused by PTCs.

Therapeutic suppression of premature termination codons: mechanisms and clinical considerations (review).

An estimated one-third of genetic disorders are the result of mutations that generate premature termination codons (PTCs) within protein coding genes. These disorders are phenotypically diverse and consist of diseases that affect both young and old individuals. Various small molecules have been identified that are capable of modulating the efficiency of translation termination, including select antibiotics of the aminoglycoside family and multiple novel synthetic molecules, including PTC124. Several of these agents have proved their effectiveness at promoting nonsense suppression in preclinical animal models, as well as in clinical trials. In addition, it has recently been shown that box H/ACA RNA-guided peudouridylation, when directed to modify PTCs, can also promote nonsense suppression. In this review, we summarize our current understanding of eukaryotic translation termination and discuss various methods for promoting the read-through of disease-causing PTCs, as well as the current obstacles that stand in the way of using the discussed agents broadly in clinical practice.

Nonsense-mediated decay targets have multiple sequence-related features that can inhibit translation.

Nonsense-mediated mRNA decay (NMD) is a surveillance system that eliminates transcripts with premature termination codons. In this study, we show that mRNAs targeted by NMD are also suppressed at the translational level. The low translational efficiency (TE) is a consequence of multiple features acting in concert, including low translation initiation rate, mediated by 5' secondary structure and by use of weak initiation sites, and low translation elongation speed, mediated by low codon usage bias. Despite low elongation rates, NMD transcripts show low ribosome density in the coding sequence, probably owing to low initiation rates, high abortion rates or rapid transit of the ribosome following initiation failure. The low TE is observed in the absence of NMD and is not explained by low transcript abundance. Translational inefficiency is flexible, such that NMD targets have increased TE upon starvation. We propose that the low TE predisposes to NMD and/or that it is part of a mechanism for regulation of NMD transcripts.

Multiple RNA surveillance mechanisms cooperate to reduce the amount of nonfunctional Ig kappa transcripts.

Random V(D)J junctions ensure that the diversity of the Ig primary repertoire is adapted to the vast heterogeneity of Ags. In two-thirds of cases, recombination between variable segments induces a frameshift in the open reading frame and generates a premature termination codon. In B cells harboring biallelic V(D)J rearrangement of Ig genes, transcription is known to occur on both the functional and nonfunctional alleles, generating considerable amounts of primary transcripts with out-of-frame V regions. In this study, we analyzed in cell lines and primary B cells the RNA surveillance of nonfunctional Igkappa transcripts arising from nonproductive rearrangement. We demonstrated that splicing inhibition, nonsense-mediated decay and nonsense-altered splicing each have an individual partial effect that together associate into an efficient surveillance machinery, downregulating nonfunctional Igkappa mRNA. Moreover, we provide evidence that the RNA surveillance efficiency increases throughout B cell development. Whereas splicing inhibition remains constant in most cell lines, differences in nonsense-mediated decay and nonsense-altered splicing are responsible for the higher RNA surveillance observed in plasma cells. Altogether, these data show that nonfunctionally rearranged alleles are subjected to active transcription but that multiple RNA surveillance mechanisms eradicate up to 90% of out-of-frame Igkappa mRNA.

Safety, tolerability, and pharmacokinetics of PTC124, a nonaminoglycoside nonsense mutation suppressor, following single- and multiple-dose administration to healthy male and female adult volunteers.

Nonsense (premature stop codon) mutations are causative in 5% to 15% of patients with monogenetic inherited disorders. PTC124, a 284-Dalton 1,2,4-oxadiazole, promotes ribosomal readthrough of premature stop codons in mRNA and offers therapeutic potential for multiple genetic diseases. The authors conducted 2 phase I studies of PTC124 in 62 healthy adult volunteers. The initial, single-dose study evaluated doses of 3 to 200 mg/kg and assessed fed-fasting status on pharmacokinetics following a dose of 50 mg/kg. The subsequent multiple-dose study evaluated doses from 10 to 50 mg/kg/dose twice per day (bid) for up to 14 days. PTC124 administered orally as a liquid suspension was palatable and well tolerated through single doses of 100 mg/kg. At 150 and 200 mg/kg, PTC124 induced mild headache, dizziness, and gastrointestinal events. With repeated doses through 50 mg/kg/dose bid, reversible transaminase elevations <2 times the upper limit of normal were sometimes observed. Immunoblot analyses of peripheral blood mononuclear cell extracts revealed no protein elongation due to nonspecific ribosomal readthrough of normal stop codons. PTC124 plasma concentrations exceeding the 2- to 10-microg/mL values associated with activity in preclinical genetic disease models were safely achieved. No sex-related differences in pharmacokinetics were seen. No drug accumulation with repeated dosing was apparent. Diurnal variation was observed, with greater PTC124 exposures after evening doses. PTC124 excretion in the urine was <2%. PTC124 pharmacokinetics were described by a 1-compartment model. Collectively, the data support initiation of phase II studies of PTC124 in patients with nonsense mutation-mediated cystic fibrosis and Duchenne muscular dystrophy.

Inhibition of nonsense-mediated mRNA decay rescues the phenotype in Ullrich's disease.

Nonsense-mediated mRNA decay (NMD) is an mRNA surveillance system that eliminates aberrant mRNAs containing premature translation termination codons (PTCs). We evaluated the role of NMD in of Ullrich's disease. The patient has a frameshift mutation with a PTC in the collagen VI alpha2 gene causing the loss of collagen VI and functional defects in extracellular matrix (ECM). The pharmacological block of NMD caused upregulation of the mutant collagen VI alpha2 subunit, resulting in collagen VI assembly and partially functional ECM formation. Our results suggest that NMD inhibitors can be used as a therapeutic tool to rescue some human genetic diseases exacerbated by NMD.