TLN468 changes the pattern of tRNA used to read through premature termination codons in CFTR.

Nonsense mutations account for 12 % of cystic fibrosis (CF) cases. The presence of a premature termination codon (PTC) leads to gene inactivation, which can be countered by the use of drugs stimulating PTC readthrough, restoring production of the full-length protein. We recently identified a new readthrough inducer, TLN468, more efficient than gentamicin. We measured the readthrough induced by these two drugs with different cystic fibrosis transmembrane conductance regulator (CFTR) PTCs. We then determined the amino acids inserted at the S1196X, G542X, W846X and E1417X PTCs of CFTR during readthrough induced by gentamicin or TLN468. TLN468 significantly promoted the incorporation of one specific amino acid, whereas gentamicin did not greatly modify the proportions of the various amino acids incorporated relative to basal conditions. The function of the engineered missense CFTR channels corresponding to these four PTCs was assessed with and without potentiator. For the recoded CFTR, except for E1417Q and G542W, the PTC readthrough induced by TLN468 allowed the expression of CFTR variants that were correctly processed and had significant activity that was enhanced by CFTR modulators. These results suggest that it would be relevant to assess the therapeutic benefit of TLN468 PTC suppression in combination with CFTR modulators in preclinical assays.

2-Guanidino-quinazoline promotes the readthrough of nonsense mutations underlying human genetic diseases.

Premature termination codons (PTCs) account for 10 to 20% of genetic diseases in humans. The gene inactivation resulting from PTCs can be counteracted by the use of drugs stimulating PTC readthrough, thereby restoring production of the full-length protein. However, a greater chemical variety of readthrough inducers is required to broaden the medical applications of this therapeutic strategy. In this study, we developed a reporter cell line and performed high-throughput screening (HTS) to identify potential readthrough inducers. After three successive assays, we isolated 2-guanidino-quinazoline (TLN468). We assessed the clinical potential of this drug as a potent readthrough inducer on the 40 PTCs most frequently responsible for Duchenne muscular dystrophy (DMD). We found that TLN468 was more efficient than gentamicin, and acted on a broader range of sequences, without inducing the readthrough of normal stop codons (TC).

Factors influencing readthrough therapy for frequent cystic fibrosis premature termination codons.

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. Nonfunctional 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.