2000 Wilschanski M, Famani C, Blau H, Rivlin J, Augarten A, Vital A, Kerem B, Kerem E. A pilot study of the effect of gentamicin on nasal potential difference measurements in cystic fibrosis patients carrying stop mutations. Am J Resp Crit Care 2000; 161:860-865. [PubMed]
This study, by Michael Wilschanski and colleagues from Haddash University Hospital, Israel, was the first to determine if gentamicin in vivo can activate mutant CFTR in CF patients carrying stop mutations as had been suggested by Howard et al, (1996) and Bedwell et al, (1997) (both abstracts below).
Nine people with CF carrying stop mutations received gentamicin nasal drops for 14 days. The abnormal nasal potential difference improved after the gentamicin treatment suggesting that chloride transport had increased. The authors concluded that gentamicin may influence the underlying chloride transport abnormality in patients with CF carrying stop mutations (i.e. those mutations containing an X).
Aminoglycoside antibiotics can apparently increase the frequency of erroneous insertion of nonsense codons hence permitting the translation of CFTR alleles carrying missense mutations to continue reading to the end of the gene. It is appropriate that this study came from Israel as 64% of people with CF that country have at least one stop mutation.
– Previously Howard and co-workers had demonstrated in cells carrying CFTR nonsense mutations, that gentamicin induced a dose-dependent increase in expression of full-length CFTR. (Howard M, Frizzell RA, Bedwell DM. Aminoglycoside antibiotics restore CFTR function by overcoming premature stop mutations. Nature Med 1996; 2:467-469. [PubMed]). Subsequently, Bedwell and co-workers showed in a CF bronchial epithelial cell line carrying the CFTR W1282X premature stop mutation, that gentamicin was capable of restoring CFTR expression on the apical membrane (Bedwell DM, Keanjak A, Bebok Z, Bubien JK, Tousson A, Clancy JP, Sorscher EJ. Suppression of a CFTR premature stop mutation in a bronchial epithelial cell line. Nature Med 1997; 3:1280-1284. [PubMed]). Intravenous gentamicin was also capable of producing small increases in CFTR conductance as judged by nasal PD measurements.
(Clancy JP, Bebok Z, Ruiz F, King C Jones J, Walker L, Hong J, Wing L, Macaluso M, Lyrene R, Sorscher EJ, Bedwell DM. Evidence that systemic gentamicin suppresses premature stop mutations in patients with cystic fibrosis. Am J Resp Crit Care 2001; 163: 1683-1692.[PubMed]).
Further work from Israel on gentamicin and stop mutations.
2003 Wilschanski M, Yahav Y, Yaacov Y, Blau H, Bentur L, Rivlin J. Aviram M, Bdolah-Abram T, Bebok Z, Shushi L, Kerem B, Kerem E. Gentamicin induced correction of CFTR function in patients with cystic fibrosis and CFTR stop mutations. N Eng J Med 2003; 349:1433-41. [PubMed].
In a double-blind, placebo-controlled, crossover trial, patients with stop mutations in CFTR or patients homozygous for the DeltaF508 mutation received nasal gentamicin drops or placebo for two consecutive periods of 14 days. The gentamicin treatment caused a significant reduction in basal nasal potential difference in the 19 patients carrying one or two stop mutations (from -45 (+/-8) to -34 (+/-11) mV, P=0.005) and a significant response to chloride-free isoproterenol solution (from 0 (+/-3.6) to -5 (+/-2.7) mV, P<0.001). Also after gentamicin treatment, there was a significant increase in peripheral and surface staining for CFTR in the nasal epithelial cells of the patients carrying stop mutations.
– So in patients with CF, who have premature stop codons, gentamicin was confirmed as causing translational “read through,” resulting in the expression of full-length CFTR protein at the apical cell membrane, and corrected towards normal the typical electrophysiological abnormalities caused by CFTR dysfunction. Subsequently another compound, PTC 124, seemed to do the job more efficiently and went into clinical trials. Identified as PTC 124 (later Ataluren), the new chemical entity selectively induces ribosomal read through of premature but not normal termination codons.
2007 Welch EM, Barton ER, Zhuo J, Tomizawa Y, Friesen WJ, Trifillis P, Paushkin S, Patel M, Trotta CR, Hwang S, Wilde RG, Karp G, Takasugi J, Chen G, Jones S, Ren H, Moon YC, Corson D, Turpoff AA, Campbell JA, Conn MM, Khan A, Almstead NG, Hedrick J, Mollin A, Risher N, Weetall M, Yeh S, Branstrom AA, Colacino JM, Babiak J, Ju WD, Hirawat S, Northcutt VJ, Miller LL, Spatrick P, He F, Kawana M, Feng H, Jacobson A, Peltz SW, Sweeney HL. PTC124 targets genetic disorders caused by nonsense mutations. Nature 2007; 447 (7140):87-91. [PubMed]
PTC124 selectively induces ribosomal readthrough of premature but not normal termination codons. PTC124 activity 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. The drug may have broad clinical potential for the treatment of a large group of genetic disorders with limited or no therapeutic options including muscular dystrophy and cystic fibrosis.
2007 Linde L, Boelz S, Nissim-Rafinia M, Oren YS, Wilschanski M, Yaacov Y, Virgilis D, Neu-Yilik G, Kulozik AE, Kerem E, Kerem B. Nonsense-mediated mRNA decay affects nonsense transcript levels and governs response of cystic fibrosis patients to gentamicin. J Clin Invest 2007; 117:683-692. [PubMed].
Aminoglycosides can readthrough premature termination codons (PTCs), permitting translation of full-length proteins. Previously the authors have found variable efficiency of readthrough in response to the aminoglycoside gentamicin among cystic fibrosis (CF) patients, all carrying the W1282X nonsense mutation. Here the authors demonstrate that there are patients in whom the level of CF transmembrane conductance regulator (CFTR) nonsense transcripts is markedly reduced, while in others it is significantly higher. Response to gentamicin was found only in patients with the higher level. They further investigated the possibility that the nonsense-mediated mRNA decay (NMD) might vary among cells and hence governs the level of nonsense transcripts available for readthrough. Their results demonstrate differences in NMD efficiency of CFTR transcripts carrying the W1282X mutation among different epithelial cell lines derived from the same tissue. Variability was also found for 5 physiologic NMD substrates, RPL3, SC35 1.6 kb, SC35 1.7 kb, ASNS, and CARS.
Importantly, their results demonstrate the existence of cells in which NMD of all transcripts was efficient and others in which the NMD was less efficient. Downregulation of NMD in cells carrying the W1282X mutation increased the level of CFTR nonsense transcripts and enhanced the CFTR chloride channel activity in response to gentamicin.
The authors suggest that their results suggest that the efficiency of NMD might vary and hence have an important role in governing the response to treatments aiming to promote readthrough of PTCs in many genetic diseases.
2007 Clancy JP, Rowe SM, Bebok Z, Aitken ML, Gibson R, Zeitlin P, Berclaz P, Moss R, Knowles MR, Oster RA, Mayer-Hamblett N, Ramsey B. No detectable improvements in cystic fibrosis transmembrane conductance regulator by nasal aminoglycosides in patients with cystic fibrosis with stop mutations. Am J Resp Cell Mol 2007; 37:57-66. [PubMed].
A US multicenter study was conducted in two cohorts of patients with CF, those heterozygous for stop mutations in the CFTR gene and those without nonsense mutations, to investigate the effects of both gentamicin and tobramycin administered over a 28-day period on sequential nasal potential difference and airway cell immunofluorescence endpoints. Eleven patients with CF who had stop mutations were enrolled in a randomized, double-blinded, crossover fashion to receive each drug, while 18 subjects with CF without stop mutations were randomized 1:1 in a parallel fashion to receive one drug. After demonstration of drug delivery, neither aminoglycoside produced detectable changes in nasal ion transport or CFTR localization in brushed cells from either study group. These results with first-generation suppressive agents suggest the need for improved drug delivery methods and/or more potent suppressors of nonsense mutations to confer CFTR correction in subjects with CF heterozygous for nonsense mutations. The study provides valuable information on parameters of the nasal potential difference measurements for use in future multicenter clinical trials.
– The results appear to conflict with earlier work of Wilschanski et al (2000 and 2003 above) but variability of response has been attributed to the efficiency of nonsense-mediated mRNA decay which may vary and hence have an important role in governing the response to treatments aiming to suppress nonsense mutations. (2007 Linde L, et al. Nonsense-mediated mRNA decay affects nonsense transcript levels and governs response of cystic fibrosis patients to gentamicin. J Clin Invest 2007; 117:683-692.[PubMed]).
2007 Rowe SM, Varga K, Rab A, Bebok Z, Byram K, Li Y, Sorscher EJ, Clancy JP. Restoration of W1282X CFTR activity by enhanced expression. Am J Resp Cell Mol Biol 2007; 37:347-356. [PubMed]
Various aminoglycosides induce “translational readthrough” of premature stop codons and have been shown to restore full-length functional protein in a number of preclinical and clinical settings. The authors studied two well-described premature termination codons found in the distal open reading frame of CFTR, W1282X and R1162X, expressed in polarizing and non-polarizing cells.
Their findings indicate that W1282X CFTR-expressing cells demonstrate significantly greater CFTR activity when over-expressed compared with R1162X CFTR cells, even when truncated protein is the predominant form. In addition, their results show that the combination of stimulated expression and stop codon suppression produces additive effects on CFTR-mediated ion transport.
– These findings are considered to provide evidence that W1282X CFTR exhibits membrane localization and retained chloride channel function after enhanced expression, and suggest that patients harbouring this mutation may be more susceptible to CFTR rescue.
2007 Sermet-Gaudelus I, Renouil M, Fajac A, Bidou L, Parbaille B, Pierrot S, Davy N, Bismuth E, Reinert P, Lenoir G. Les BMC Medicine 2007; 5:5. In vitro prediction of stop-codon suppression by intravenous gentamicin in patients with cystic fibrosis: a pilot study. [PubMed]
A pilot study was conducted to determine whether intravenous gentamicin suppresses stop codons in CF patients and whether it has clinical benefits. A dual gene reporter system was used to determine the gentamicin-induced readthrough level of the most frequent stop mutations within the CFTR in the French population. The authors investigated readthrough efficiency in response to 10 mg/kg once-daily intravenous gentamicin perfusions in patients with and without stop mutations. Respiratory function, sweat chloride concentration, nasal potential difference (NPD) and CFTR expression in nasal epithelial cells were measured at baseline and after 15 days of treatment.
After in vitro gentamicin incubation, the readthrough efficiency for the Y122X mutation was at least five times higher than that for G542X, R1162X, and W1282X. In six of the nine patients with the Y122X mutation, CFTR immunodetection showed protein at the membrane of the nasal epithelial cells and the CFTR-dependent Cl- secretion in NPD measurements increased significantly. Respiratory status also improved in these patients, irrespective of the gentamicin sensitivity of the bacteria present in the sputum. Mean sweat chloride concentration decreased significantly and normalised in two patients. Clinical status, NPD and sweat Cl- values did not change in the Y122X patients with no protein expression, in patients with the other stop mutations investigated in vitro and those without stop mutations.
The authors concluded suppression of stop mutations in the CFTR gene with parenteral gentamicin can be predicted in vitro and is associated with clinical benefit and significant modification of the CFTR-mediated Cl- transport in nasal and sweat gland epithelium.
2008 Kerem E, Hirawat S, Armoni S, Yaakov Y, Shoseyov D, Cohen M, Nissim-Rafinia M, Blau H, Rivlin J, Aviram M, Elfring GL, Northcutt VJ, Miller LL, Kerem B, Wilschanski M. Effectiveness of PTC124 treatment of cystic fibrosis caused by nonsense mutations: a prospective phase II trial. Lancet 2008; 372:719-727. [PubMed]
PTC124 is an orally bioavailable small molecule that is designed to induce ribosomes to selectively read through premature stop codons during mRNA translation, to produce functional CFTR: This phase II prospective trial recruited adults with cystic fibrosis who had at least one nonsense mutation in the CFTR gene. Patients were assessed in two 28-day cycles. During the first cycle, patients received PTC124 at 16 mg/kg per day in three doses every day for 14 days, followed by 14 days without treatment; in the second cycle, patients received 40 mg/kg of PTC124 in three doses every day for 14 days, followed by 14 days without treatment.
The primary outcome had three components: change in CFTR-mediated total chloride transport; proportion of patients who responded to treatment; and normalisation of chloride transport, as assessed by transepithelial nasal potential difference (PD) at baseline, at the end of each 14-day treatment course, and after 14 days without treatment. Transepithelial nasal PD was evaluated in 23 patients in the first cycle and in 21 patients in the second cycle.
Mean total chloride transport increased in the first treatment phase, with a change of -7.1 (SD 7.0) mV (p<0.0001), and in the second, with a change of -3.7 (SD 7.3) mV (p=0.032). We recorded a response in total chloride transport (defined as a change in nasal PD of -5 mV or more) in 16 of the 23 patients in the first cycle’s treatment phase (p<0.0001) and in eight of the 21 patients in the second cycle (p<0.0001). Total chloride transport entered the normal range for 13 of 23 patients in the first cycle’s treatment phase (p=0.0003) and for nine of 21 in the second cycle (p=0.02). Two patients given PTC124 had constipation without intestinal obstruction, and four had mild dysuria. No drug-related serious adverse events were recorded.
The authors concluded that in patients with cystic fibrosis who have a premature stop codon in the CFTR gene, oral administration of PTC124 to suppress nonsense mutations reduces the epithelial electrophysiological abnormalities caused by CFTR dysfunction.
2010 Sermet-Gaudelius I, Boeck KD, Casimir GJ, Vermeulen F, leal T, Mogegnet A, Roussel D, Fritch J, hanssens L, Hirawat S, Miller NL, Constantine S, Reha A, Ajayi T, Elfring GL, Miller LL. Ataluren (PTC124) induces cystic fibrosis transmembrane conductance regulator protein expression and activity in children with nonsense mutation cystic fibrosis. Am J Respir Crit Care 2010; 182:1262-1272.[PubMed]
A study to evaluate ataluren activity, safety, and pharmacokinetics in children with nonsense mutation CF. Patients were assessed in two 28-day cycles, comprising 14 days on and 14 days off ataluren. Patients took ataluren three times per day (morning, midday, and evening) with randomization to the order of receiving a lower dose (4, 4, and 8 mg/kg) and a higher dose (10, 10, and 20 mg/kg) in the two cycles. The study enrolled 30 patients (16 male and 14 female, ages 6 through 18 yr) with a nonsense mutation in at least one allele of the CFTR gene, a classical CF phenotype, and abnormal baseline nasal epithelial chloride transport.
Ataluren induced a nasal chloride transport response (at least a -5-mV improvement) or hyperpolarization (value more electrically negative than -5 mV) in 50% and 47% of patients, respectively, with more hyperpolarizations at the higher dose. Improvements were seen in seven of nine nonsense mutation genotypes represented. Ataluren significantly increased the proportion of nasal epithelial cells expressing apical full-length CFTR protein. Adverse events and laboratory abnormalities were infrequent and usually mild. Ataluren pharmacokinetics were similar to those in adults.
The authors concluded that in children with nonsense mutations CF, Ataluren can induce functional CFTR production and is well tolerated.
2011 Wilschanski M, Miller LL, Shoseyov D, Blau H, Rivlin J, Aviram M, Cohen M, Armoni S, Yaakov Y, Pugatch T, Cohen-Cymberknoh M, Miller NL, Reha A, Northcutt VJ, Hirawat S, Donnelly K, Elfring GL, Ajayi T, Kerem E. Chronic ataluren (PTC124) treatment of nonsense mutation cystic fibrosis. Eur Respir J 2011; 38:59-69. [PubMed]
Ataluren (PTC124) allows ribosomal read through of premature stop codons in mRNA. The authors evaluated drug activity and safety in patients with nonsense CF mutations who took ataluren three times daily for 12 weeks at either a lower dose (4, 4 and 8 mg.kg(-1)) or higher dose (10, 10 and 20 mg.kg(-1)). The study enrolled 19 patients (10 males and nine females aged 19-57 yrs; dose: lower 12, higher seven) with a classic CF phenotype, at least one CFTR nonsense mutation allele, and an abnormal nasal total chloride transport. Both ataluren doses were similarly active, improving total chloride transport with a combined mean change of -5.4 mV (p<0.001), and on-treatment responses (at least -5 mV improvement) and hyperpolarisations (values more electrically negative than -5 mV) in 61% (p<0.001) and 56% (p = 0.002) of patients. CFTR function was greater with time and was accompanied by trends toward improvements in pulmonary function and CF-related coughing. Adverse clinical and laboratory findings were uncommon and usually mild. Chronic ataluren administration produced time-dependent improvements in CFTR activity and clinical parameters with generally good tolerability.
– Apparently the results in Duchenne muscular dystrophy have been disappointing and the benefits in CF were less evident in one study. However, further results in cystic fibrosis are awaited.
In June 2011 PTC therapeutics announced the results from a large multicentre Phase 3 study of ataluren (This section taken directly from http://ptct.client.share).(full reference below)
The data was presented at the ECFS Dublin Conference in 2012 by Michael Konstan.
This Phase 3 study, which was conducted across 11 countries, was a double-blind, placebo-controlled study comparing ataluren (n=116) to placebo (n=116) in CF patients. The primary endpoint, the relative change from baseline in %-predicted FEV1 at 48 weeks, showed a positive trend favoring ataluren versus placebo, and a larger effect in the patients not receiving chronic inhaled antibiotics. The effect of inhaled antibiotics was largely attributable to the use of inhaled aminoglycosides. In the intent-to-treat population, there was a 3% difference in the relative change from baseline in %-predicted FEV1 between the ataluren and placebo groups at Week 48 (-2.5% change on ataluren vs. -5.5% change on placebo; p=0.124). An analysis of the relative change from baseline in %-predicted FEV1 across all post-baseline study visits demonstrated an average difference between ataluren and placebo of 2.5% (-1.8% average change on ataluren vs. -4.3% average change on placebo; p= 0.0478)
The study was stratified by age, baseline FEV1, and the use of chronic inhaled antibiotics. A statistically significant effect (p=0.0072) was seen between treatment and use of inhaled antibiotics at baseline, indicating that inhaled antibiotics was a significant confounder of the overall results. A substantial treatment effect was seen in the patients not receiving chronic inhaled antibiotics at baseline; the Week 48 difference between the ataluren and placebo arms in FEV1 was 6.7% (-0.2% change on ataluren vs. -6.9% change on placebo). The secondary endpoint, the rate of pulmonary exacerbations (ie, the number of pulmonary exacerbations in 48 weeks) also showed a positive trend in favor of ataluren, with the rate in the ataluren group being 23% lower than the placebo group (p=0.0992). In the patients not receiving chronic inhaled antibiotics, the pulmonary exacerbation rate in the ataluren group was 43% lower than the rate in the placebo group. These results show a consistent treatment effect of ataluren on both pulmonary function and exacerbation rates. In patients not also on inhaled antibiotics there was a 43% reduction in pulmonary exacerbations.
In 2012 the FDA and the European Commission granted Ataluren Orphan Drug status for the treatment of nonsense mutation cystic fibrosis and nonsense mutation Duchenne and Becker muscular dystrophy.
2014 Kerem E, Konstan MW, De Boeck K, Accurso FJ, Sermet-Gaudelus I, Wilschanski M, Elborn JS, Melotti P, Bronsveld I, Fajac I, Malfroot A, Rosenbluth DB, Walker PA, McColley SA, Knoop C, Quattrucci S, Rietschel E, Zeitlin P, Barth J, Elfring GL, Welch EM, Branstrom A, Spiegel RJ, Peltz SW, Ajayi T, Rowe SM; for the Cystic Fibrosis Ataluren Study Group.Ataluren for the treatment of nonsense-mutation cystic fibrosis: a randomised, double-blind, placebo-controlled phase 3 trial. Lancet Respir Med. 2014 May 15. pii: S2213-2600(14)70100-6. doi: 10.1016/S2213-2600(14)70100-6. [Epub ahead of print][PubMed]
This is the published summary of the Phase III study described above and the conclusions are the same
– Although ataluren did not improve lung function in the overall population of nonsense-mutation cystic fibrosis patients who received this treatment, it might be beneficial for patients not taking chronic inhaled tobramycin – an effect noted in a post-hoc analysis.
There has been some dispute as to how the drug was tested questioning whether PTC 124 did actually promote read through of stop mutations (Auld DS et al. Proc Natl Acad Sci USA 2010;107:4878-4883. [PubMed] fhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC2841876/pdf/pnas.0909141107.pdf; Thorne N et al. Chem Biol 2010; 17:646-657. [PubMed]http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2925662/pdf/nihms217027.pdf). These suggestions were disputed (Peltz et al. Proc Nat Acad Sci USA 2009;106:E64http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2700894/pdf/zpqe64.pdf) although a second study testing PTC 124 across a range of assays found no evidence of read through McElroy et al. PLos Biol 2013;11:e1001593http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3692445/pdf/pbio.1001593.pdf) and problems relating to firefly luciferase used in the assay were discussed (Roberts RG. PLos Biol 2013;11:e1001458http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3692443/pdf/pbio.1001458.pdf
(This account based in part on “Ataluran: reading through the controveries”. Courtesy CFNN (Cystic Fibrosis News Network), July 8,2014 – with permission)
2014 Kerem E. Konstan MW. De Boeck K. Accurso FJ. Sermet-Gaudelus I. Wilschanski M. Elborn JS. Melotti P. Bronsveld I. Fajac I. Malfroot A. Rosenbluth DB. Walker PA. McColley SA. Knoop C. Quattrucci S. Rietschel E. Zeitlin PL. Barth J. Elfring GL. Welch EM. Branstrom A. Spiegel RJ. Peltz SW. Ajayi T. Rowe SM. Cystic Fibrosis Ataluren Study Group. Ataluren for the treatment of nonsense-mutation cystic fibrosis: a randomised, double-blind, placebo-controlled phase 3 trial. Lancet Respir Med 2014; 2(7):539-47.[PubMed]
A randomised, double-blind, placebo-controlled, phase 3 study enrolled patients from 36 sites in 11 countries in North America and Europe. Eligible patients with nonsense-mutation cystic fibrosis (aged > 6 years; abnormal nasal potential difference; sweat chloride >40 mmol/L; forced expiratory volume in 1 s [FEV1] > 40% and < 90%) were randomly assigned by interactive response technology to receive oral ataluren (10 mg/kg in morning, 10 mg/kg midday, and 20 mg/kg in evening) or matching placebo for 48 weeks. Randomisation used a block size of four, stratified by age, chronic inhaled antibiotic use, and percent-predicted FEV1. The primary endpoint was relative change in percent-predicted FEV1 from baseline to week 48, analysed in all patients with a post-baseline spirometry measurement. This study is registered with ClinicalTrials.gov, number NCT00803205.
Between Sept 8, 2009, and Nov 30, 2010, 238 patients were randomly assigned, of whom 116 in each treatment group had a valid post-baseline spirometry measurement. Relative change from baseline in percent-predicted FEV1 did not differ significantly between ataluren and placebo at week 48 (-2.5% vs -5.5%; difference 3.0% [95% CI -0.8 to 6.3]; p=0.12). The number of pulmonary exacerbations did not differ significantly between treatment groups (rate ratio 0.77 [95% CI 0.57-1.05]; p=0.0992).
However, post-hoc analysis of the subgroup of patients not using chronic inhaled tobramycin showed a 5.7% difference (95% CI 1.5-10.1) in relative change from baseline in percent-predicted FEV1 between the ataluren and placebo groups at week 48 (-0.7% [-4.0 to 2.1] vs -6.4% [-9.8 to -3.7]; nominal p=0.0082), and fewer pulmonary exacerbations in the ataluren group (1.42 events [0.9-1.9] vs 2.18 events [1.6-2.7]; rate ratio 0.60 [0.42-0.86]; nominal p=0.0061). Safety profiles were generally similar for ataluren and placebo, except for the occurrence of increased creatinine concentrations (ie, acute kidney injury), which occurred in 18 (15%) of 118 patients in the ataluren group compared with one (<1%) of 120 patients in the placebo group. No life-threatening adverse events or deaths were reported in either group.
The authors concluded that although ataluren did not improve lung function in the overall population of nonsense-mutation cystic fibrosis patients who received this treatment, but it might be beneficial for patients not taking chronic inhaled tobramycin.
– As mentioned above there has been some difference of opinion as to the action of ataluren and as to whether the drug actually does promote read through of nonsense mutations. It has been suggested that the assay using luciferase as the reporter gene when positive, rather than indicating transcription, merely indicates direct stimulation of the reporter gene.
2014 Lentini L. Melfi R. Di Leonardo A. Spinello A. Barone G. Pace A. Palumbo Piccionello A. Pibiri I. Toward a rationale for the PTC124 (Ataluren) promoted readthrough of premature stop codons: a computational approach and GFP-reporter cell-based assay. Mol Pharm 2014; 11(3):653-64. [PubMed]
The presence in the mRNA of premature stop codons (PTCs) results in protein truncation responsible for several inherited (genetic) diseases. A well-known example of these diseases is cystic fibrosis, where approximately 10% (worldwide) of patients have nonsense mutations in the CF transmembrane regulator (CFTR) gene. PTC124 (3-(5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl)-benzoic acid), also known as Ataluren, is a small molecule that has been suggested to allow PTC read through even though its target has yet to be identified.
In the lack of a general consensus about its mechanism of action, the authors experimentally tested the ability of PTC124 to promote the read through of premature termination codons by using a new reporter. The reporter vector was based on a plasmid harbouring the H2B histone coding sequence fused in frame with the green fluorescent protein (GFP) cDNA, and a TGA stop codon was introduced in the H2B-GFP gene by site-directed mutagenesis. Additionally, an unprecedented computational study on the putative supramolecular interaction between PTC124 and an 11-codon (33-nucleotides) sequence corresponding to a CFTR mRNA fragment containing a central UGA nonsense mutation showed a specific interaction between PTC124 and the UGA codon.
– Apparently the conclusion is that, the H2B-GFP-opal based assay and the molecular dynamics (MD) simulation support the hypothesis that PTC124 is able to promote the specific read through of internal TGA premature stop codons.
2016 Shoseyov D, Cohen-Cymberknoh M, Wilschanski M. Ataluren for the treatment of cystic fibrosis. Expert Rev Respir Med. 2016 Feb 24:1-5. [Epub ahead of print] [PubMed]
Ataluren is orally bioavailable and was shown to be effective in Cystic Fibrosis. Phase I and II studies established the safety and dosing regimens for Ataluren. The results of a short study showed modest improvements in pulmonary function and a reduction in quantitative cough assessment. There was improvement in nasal potential difference and nasal epithelial CFTR protein. In a phase III trial this effect was not observed in patients that were concomitantly treated with tobramycin inhalation. Following these positive findings, a multinational Phase III placebo-controlled efficacy trial is currently underway.
– This paper sums up the present situation regarding the use of ataluren for people with CF and stop mutations.
Dr David Shoseyov (figure) is Senior Physician and Advisor on Pediatric Lung Disease, Hadassah Medical Center, Jerusalem and Chair of Israeli Cystic Fibrosis Council’s Medical Advisory Committee
2017 Aslam AA, Higgins C, Sinha IP, Southern KW. Ataluren and similar compounds (specific therapies for premature termination codon class I mutations) for cystic fibrosis. Cochrane Database Syst Rev. 2017 Jan 19;1:CD012040. doi: 10.1002/14651858.CD012040.pub2. [Pubmed]
Objectives were to evaluate the benefits and harms of ataluren and similar compounds on clinically important outcomes in people with cystic fibrosis with class I mutations (premature termination codons). Searches identified 28 references to eight trials; five trials were excluded.
The authors concluded there is currently insufficient evidence to determine the effect of ataluren as a therapy for people with cystic fibrosis with class I mutations. Future trials should carefully assess for adverse events, notably renal impairment and consider the possibility of drug interactions. Cross-over trials should be avoided given the potential for the treatment to change the natural history of cystic fibrosis.
~A useful review on the present state of this treatment of these class 1 mutations.
2018 Pranke I, Bidou L, Martin N, Blanchet S, Hatton A, Karri S, Cornu D, Costes B, Chevalier B, Tondelier D, Girodon E, Coupet M, Edelman A, Fanen P, Namy O, Sermet-Gaudelus I, Hinzpeter A. Factors influencing readthrough therapy for frequent cystic fibrosis premature termination codons. ERJ Open Res.2018 Feb 23;4(1). pii: 00080-2017. doi: 10.1183/23120541.00080-2017. eCollection 2018 Jan.Full text available. [PubMed]
Premature termination codons (PTCs) are generally associated with severe forms of genetic diseases. Readthrough of in-frame PTCs using small molecules is a promising therapeutic approach. Nonetheless, the outcome of preclinical studies has been low and variable. Treatment efficacy depends on: 1) the level of drug-induced readthrough, 2) the amount of target transcripts, and 3) the activity of the recoded protein.
The aim of the present study was to identify, in the cystic fibrosis transmembrane conductance regulator (CFTR) model, recoded channels from readthrough therapy that may be enhanced using CFTR modulators. First, drug-induced readthrough of 15 PTCs was measured using a dual reporter system under basal conditions and in response to gentamicin and negamycin. Secondly, exon skipping associated with these PTCs was evaluated with a minigene system. Finally, incorporated amino acids were identified by mass spectrometry and the function of the predicted recoded CFTR channels corresponding to these 15 PTCs was measured. Non-functional channels were subjected to CFTR-directed ivacaftor-lumacaftor treatments. The results demonstrated that CFTR modulators increased activity of recoded channels, which could also be confirmed in cells derived from a patient. In conclusion, this work will provide a framework to adapt treatments to the patient’s genotype by identifying the most efficient molecule for each PTC and the recoded channels needing co-therapies to rescue channel function.
– A helpful review of the present situation regarding PTCs from a leading French group
Dr. Iwona Pranke is Post Doctoral Researcher at the Institut Necker, Paris
Konstan MW, VanDevanter DR, Rowe SM, Wilschanski M, Kerem E, Sermet-Gaudelus I, DiMango E, Melotti P, McIntosh J, De Boeck K; ACT CF Study Group. Efficacy and safety of ataluren in patients with nonsense-mutation cystic fibrosis not receiving chronic inhaled aminoglycosides: The international, randomized, double-blind, placebo-controlled Ataluren Confirmatory Trial in Cystic Fibrosis (ACT CF). J Cyst Fibros. 2020 Jan 23. pii: S1569-1993(20)30030-8. doi: 10.1016/j.jcf.2020.01.007. [Epub ahead of print] [Pubmed]
Ataluren was developed for potential treatment of nonsense-mutation cystic fibrosis (CF). A previous phase 3 ataluren study failed to meet its primary efficacy endpoint, but post-hoc analyses suggested that aminoglycosides may have interfered with ataluren’s action. Thus, this subsequent trial (NCT02139306) was designed to assess the efficacy and safety of ataluren in patients with nonsense-mutation CF not receiving aminoglycosides.
Eligible subjects with nonsense-mutation CF (aged ≥6 years; percent predicted (pp) FEV1 ≥40 and ≤90) from 75 sites in 16 countries were randomly assigned in double-blinded fashion to receive oral ataluren or matching placebo thrice daily for 48 weeks. The primary endpoint was absolute change in average ppFEV1 from baseline to the average of Weeks 40 and 48.
279 subjects were enrolled; 138 subjects in the ataluren arm and 136 in the placebo arm were evaluable for efficacy. Absolute ppFEV1 change from baseline did not differ significantly between the ataluren and placebo groups at Week 40 (-0.8 vs -1.8) or Week 48 (-1.7 vs -2.4). Average ppFEV1 treatment difference from baseline to Weeks 40 and 48 was 0.6 (95% CI -1.3, 2.5; p = 0.54). Pulmonary exacerbation rate per 48 weeks was not significantly different (ataluren 0.95 vs placebo 1.13; rate ratio p = 0.40). Safety was similar between groups. No life-threatening adverse events or deaths were reported.
Neither ppFEV1 change nor pulmonary exacerbation rate over 48 weeks were statistically different between ataluren and placebo groups. Development of a nonsense-mutation CF therapy remains elusive.
– Disappointing result when earlier work seemed so promising.
Dr Michael Konstan is the Gertrude Lee Chandler Tucker Professor of Pediatrics, Department of Pediatrics, School of Medicine, Case Western Reserve University
Martin Y Ng, Hong Li, Mikel D Ghelfi, Yale E Goldman, Barry S Cooperman. Ataluren and aminoglycosides stimulate read-through of nonsense codons by orthogonal mechanisms. Proc Natl Acad Sci U S A 2021 Jan 12;118(2):e2020599118.doi: 10.1073/pnas.2020599118.Free article [Pubmed]
During protein synthesis, nonsense mutations, resulting in premature stop codons (PSCs), produce truncated, inactive protein products. Such defective gene products give rise to many diseases, including cystic fibrosis, Duchenne muscular dystrophy (DMD), and some cancers.
Small molecule nonsense suppressors, known as TRIDs (translational read-through-inducing drugs), stimulate stop codon read-through. The best characterized TRIDs are ataluren, which has been approved by the European Medicines Agency for the treatment of DMD, and G418, a structurally dissimilar aminoglycoside. Previously, we applied a highly purified in vitro eukaryotic translation system to demonstrate that both aminoglycosides like G418 and more hydrophobic molecules like ataluren stimulate read-through by direct interaction with the cell’s protein synthesis machinery.
Our results suggested that they might do so by different mechanisms. Here, we pursue this suggestion through a more-detailed investigation of ataluren and G418 effects on read-through. We find that ataluren stimulation of read-through derives exclusively from its ability to inhibit release factor activity. In contrast, G418 increases functional near-cognate tRNA mispairing with a PSC, resulting from binding to its tight site on the ribosome, with little if any effect on release factor activity. The low toxicity of ataluren suggests that development of new TRIDs exclusively directed toward inhibiting termination should be a priority in combatting PSC diseases. Our results also provide rate measurements of some of the elementary steps during the eukaryotic translation elongation cycle, allowing us to determine how these rates are modified when cognate tRNA is replaced by near cognate tRNA ± TRIDs.
Martin Y Ng is at the Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104.
Sylwia Michorowska. Ataluren-Promising Therapeutic Premature Termination Codon Readthrough Frontrunner. Pharmaceuticals (Basel). 2021 Aug 9;14(8):785.doi: 10.3390/ph14080785. [Pubmed]Free PMC article
Around 12% of hereditary disease-causing mutations are in-frame nonsense mutations. The expression of genes containing nonsense mutations potentially leads to the production of truncated proteins with residual or virtually no function. However, the translation of transcripts containing premature stop codons resulting in full-length protein expression can be achieved using readthrough agents. Among them, only ataluren was approved in several countries to treat nonsense mutation Duchenne muscular dystrophy (DMD) patients.
This review summarizes ataluren’s journey from its identification, via first in vitro activity experiments, to clinical trials in DMD, cystic fibrosis, and aniridia. Additionally, data on its pharmacokinetics and mechanism of action are presented. The range of diseases with underlying nonsense mutations is described for which ataluren therapy seems to be promising. What is more, experiments in which ataluren did not show its readthrough activity are also included, and reasons for their failures are discussed.
Dr Sylwia Michorowska is in the Department of Bioanalysis and Drug Analysis, Faculty of Pharmacy, Medical University of Warsaw, 02-097 Warsaw, Poland.
– The full article is an interesting very detailed review of the development and clinical trials of ataluren, initially known as PTC124, in muscular dystrophy, cystic fibrosis and various metabolic disorders.