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A premature termination codon (PTC) in the ORF of an mRNA generally leads to production of a truncated polypeptide, accelerated degradation of the mRNA, and depression of overall mRNA expression. Accordingly, nonsense mutations cause some of the most severe forms of inherited disorders. The small-molecule drug ataluren promotes therapeutic nonsense suppression and has been thought to mediate the insertion of near-cognate tRNAs at PTCs. However, direct evidence for this activity has been lacking. Here, we expressed multiple nonsense mutation reporters in human cells and yeast and identified the amino acids inserted when a PTC occupies the ribosomal A site in control, ataluren-treated, and aminoglycoside-treated cells. We find that ataluren’s likely target is the ribosome and that it produces full-length protein by promoting insertion of near-cognate tRNAs at the site of the nonsense codon without apparent effects on transcription, mRNA processing, mRNA stability, or protein stability. The resulting readthrough proteins retain function and contain amino acid replacements similar to those derived from endogenous readthrough, namely Gln, Lys, or Tyr at UAA or UAG PTCs and Trp, Arg, or Cys at UGA PTCs. These insertion biases arise primarily from mRNA:tRNA mispairing at codon positions 1 and 3 and reflect, in part, the preferred use of certain nonstandard base pairs, e.g., U-G. Ataluren’s retention of similar specificity of near-cognate tRNA insertion as occurs endogenously has important implications for its general use in therapeutic nonsense suppression.

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.

Nonsense mutations, resulting in a premature stop codon in the open reading frame of mRNAs are responsible for thousands of inherited diseases. Readthrough of premature stop codons by small molecule drugs has emerged as a promising therapeutic approach to treat disorders resulting from premature termination of translation. The aminoglycoside antibiotics are a class of molecule known to promote readthrough at premature termination codons. Gentamicin consists of a mixture of major and minor aminoglycoside components. Here, we investigated the readthrough activities of the individual components and show that each of the four major gentamicin complex components representing 92-99% of the complex each had similar potency and activity to that of the complex itself. In contrast, a minor component (gentamicin X2) was found to be the most potent and active readthrough component in the gentamicin complex. The known oto- and nephrotoxicity associated with aminoglycosides preclude long-term use as readthrough agents. Thus, we evaluated the components of the gentamicin complex as well as the so-called \"designer\" aminoglycoside, NB124, for in vitro and in vivo safety. In cells, we observed that gentamicin X2 had a safety/readthrough ratio (cytotoxicity/readthrough potency) superior to that of gentamicin, G418 or NB124. In rodents, we observed that gentamicin X2 showed a safety profile that was superior to G418 overall including reduced nephrotoxicity. These results support further investigation of gentamicin X2 as a therapeutic readthrough agent.

Ataluren promotes ribosomal readthrough of premature termination codons in mRNA which result from nonsense mutations. In vitro studies were performed to characterize the metabolism and enzyme kinetics of ataluren and its interaction potential with CYP enzymes. Incubation of [14C]‐ataluren with human liver microsomes indicated that the major metabolic pathway for ataluren is via direct glucuronidation and that the drug is not metabolized via cytochrome P450 (CYP). Glucuronidation was also observed in the incubation in human intestinal and kidney microsomes, but not in human pulmonary microsomes. UGT1A9 was found to be the major uridine diphosphate glucuronosyltransferase (UGT) responsible for ataluren glucuronidation in the liver and kidney microsomes. Enzyme kinetic analysis of the formation of ataluren acyl glucuronide, performed in human liver, kidney, and intestinal microsomes and recombinant human UGT1A9, found that increasing bovine serum albumin (BSA) levels enhanced the glucuronidation Michaelis‐Menten constant (Km) and ataluren protein binding but had a minimal effect on maximum velocity (Vmax) of glucuronidation. Due to the decreased unbound Michaelis‐Menten constant (Km,u), the ataluren unbound intrinsic clearance (CLint,u) increased for all experimental systems and BSA concentrations. Human kidney microsomes were about 3.7‐fold more active than human liver microsomes, in terms of CLint,u/mg protein, indicating that the kidney is also a key organ for the metabolism and disposition of ataluren in humans. Ataluren showed no or little potential to inhibit or induce most of the CYP enzymes.

Spinal muscular atrophy (SMA) is a genetic disease caused by mutation or deletion of the survival of motor neuron 1 (SMN1) gene. A paralogous gene in humans, SMN2, produces low, insufficient levels of functional SMN protein due to alternative splicing that truncates the transcript. The decreased levels of SMN protein lead to progressive neuromuscular degeneration and high rates of mortality. Through chemical screening and optimization, we identified orally available small molecules that shift the balance of SMN2 splicing toward the production of full-length SMN2 messenger RNA with high selectivity. Administration of these compounds to Δ7 mice, a model of severe SMA, led to an increase in SMN protein levels, improvement of motor function, and protection of the neuromuscular circuit. These compounds also extended the life span of the mice. Selective SMN2 splicing modifiers may have therapeutic potential for patients with SMA.

PTC124: a no-nonsense drug Many inherited diseases result from premature termination during translation of a messenger RNA into protein; one such disease is muscular dystrophy. Welch et al . now report that a small molecule, PTC124, enables the translation machinery to bypass sites that cause premature termination, but still terminate normally at the end of the mRNA. In human and mouse cells, this drug restores normal translation of the gene that is mutated in muscular dystrophy, and it restores muscle function in the mdx mouse model for the human disease. This work offers the hope that similar drugs might be used to target nonsense mutations and restore protein function in a wide variety of diseases. PTC124 is now undergoing clinical trials in muscular dystrophy and cystic fibrosis patients. A small molecule, PTC124, enables the translation machinery for mRNA into proteins to bypass sites that cause premature termination, but still terminate normally at the end of the mRNA. In human and mouse cells, this drug restores normal translation of the gene that is mutated in muscular dystrophy, and restores muscle function in mdx mice that model the human disease. Nonsense mutations promote premature translational termination and cause anywhere from 5–70% of the individual cases of most inherited diseases 1 . Studies on nonsense-mediated cystic fibrosis have indicated that boosting specific protein synthesis from <1% to as little as 5% of normal levels may greatly reduce the severity or eliminate the principal manifestations of disease 2 , 3 . To address the need for a drug capable of suppressing premature termination, we identified PTC124—a new chemical entity that selectively induces ribosomal readthrough of premature but not normal termination codons. PTC124 activity, optimized using nonsense-containing reporters, promoted dystrophin production in primary muscle cells from humans and mdx mice expressing dystrophin nonsense alleles, and rescued striated muscle function in mdx mice within 2–8 weeks of drug exposure. PTC124 was well tolerated in animals at plasma exposures substantially in excess of those required for nonsense suppression. The selectivity of PTC124 for premature termination codons, its well characterized activity profile, oral bioavailability and pharmacological properties indicate that this drug may have broad clinical potential for the treatment of a large group of genetic disorders with limited or no therapeutic options.

The coronavirus disease 2019 (COVID-19) pandemic has created an urgent need for therapeutics that inhibit the SARS-CoV-2 virus and suppress the fulminant inflammation characteristic of advanced illness. Here, we describe the anti-COVID-19 potential of PTC299, an orally available compound that is a potent inhibitor of dihydroorotate dehydrogenase (DHODH), the rate-limiting enzyme of the de novo pyrimidine biosynthesis pathway. In tissue culture, PTC299 manifests robust, dose-dependent, and DHODH-dependent inhibition of SARS CoV-2 replication (EC50 range, 2.0 to 31.6 nM) with a selectivity index >3,800. PTC299 also blocked replication of other RNA viruses, including Ebola virus. Consistent with known DHODH requirements for immunomodulatory cytokine production, PTC299 inhibited the production of interleukin (IL)-6, IL-17A (also called IL-17), IL-17F, and vascular endothelial growth factor (VEGF) in tissue culture models. The combination of anti-SARS-CoV-2 activity, cytokine inhibitory activity, and previously established favorable pharmacokinetic and human safety profiles render PTC299 a promising therapeutic for COVID-19. Competing Interest Statement J.L, E.M., S.B, C. S-D.-C., E.L.S., Y.W, and V.S. have no conflict of interest to declare. S.P. and R.S. received support from PTC Therapeutics for this work. J.D.G, L.C, M.W, C.T-L., N.A.N, J.M.C, M.P. E.M.W., K. O K., R.K, E.G., A.J. and S.P. are or were employed by PTC Therapeutic and have received salary compensation for time, effort, and hold or held financial interest in the company.

[...] despite the fact that aminoglycosides also promote non sense suppression3 and many patients with cystic fibrosis receive amino glycosides chroni cally by inhalation or in repeated intra venous treatments, we are unaware of any reports that patients treated with these drugs have become susceptible to novel suppression-mediated transpositions. [...] the frequency with which transposons are inactivated solely by a single nonsense mutation is quite low.4 PTC124 does not suppress mRNAs containing more than one premature nonsense codon.1 Third, almost all transposons harbouring a nonsense mutation have also accumulated additional mutations,4 presumably owing to a lack of selective pressure on these retroelements to maintain proteincoding potential.

Tauopathies are neurodegenerative diseases characterized by the abnormal accumulation of microtubule-associated protein tau (MAPT) in the brain. These disorders, like frontotemporal dementia (FTD-Tau), currently lack effective therapies and can occur sporadically or be inherited when associated with gene mutations. The gene region encompassing exon 10 and adjacent introns is a hotspot for pathogenic variants, including splicing mutations that enhance exon 10 inclusion and increase 4R tau expression, and gain-of-function mutations that generate aggregation-prone mutant 4R tau protein. For these 4R-specific tauopathies, a targeted mRNA splicing approach that promotes exon 10 exclusion may offer therapeutic benefit. In this study, we discovered novel splicing modulator compounds (SMCs) that promote exon 10 exclusion, and demonstrated their efficacy in FTD patient-derived neuronal models carrying the tau-P301L gain-of-function mutation or the tau-S305N splicing mutation. Treatment with SMC reduced 4R tau expression and decreased the accumulation of hyperphosphorylated tau (pTau), oligomeric and insoluble tau, thereby rescuing tau-associated neuronal toxicity. Importantly, our lead SMC corrected the 3R/4R splice ratio and significantly reduced pTau in the brain of a gene- replacement (GR) mouse model expressing the human tau-N279K splicing mutation. These findings support the therapeutic potential of this class of small molecules and establish pre- mRNA splicing modulation as a promising strategy for the treatment of 4R tauopathies. Discovery of SMCs that correct splicing, reduce 4R tau, and rescue pathology in patient- derived neuronal and models of 4R tauopathies.