Properties of Non-Aminoglycoside Compounds Used to Stimulate Translational Readthrough of PTC Mutations in Primary Ciliary Dyskinesia

Abstract

Primary ciliary dyskinesia (PCD) is a rare disease with autosomal recessive inheritance, caused mostly by bi-allelic gene mutations that impair motile cilia structure and function. Currently, there are no causal treatments for PCD. In many disease models, translational readthrough of premature termination codons (PTC-readthrough) induced by aminoglycosides has been proposed as an effective way of restoring functional protein expression and reducing disease symptoms. However, variable outcomes of pre-clinical trials and toxicity associated with long-term use of aminoglycosides prompt the search for other compounds that might overcome these problems. Because a high proportion of PCD-causing variants are nonsense mutations, readthrough therapies are an attractive option. We tested a group of chemical compounds with known PTC-readthrough potential (ataluren, azithromycin, tylosin, amlexanox, and the experimental compound TC007), collectively referred to as non-aminoglycosides (NAGs). We investigated their PTC-readthrough efficiency in six PTC mutations found in Polish PCD patients, in the context of cell and cilia health, and in comparison to the previously tested aminoglycosides. The NAGs did not compromise the viability of the primary nasal respiratory epithelial cells, and the ciliary beat frequency was retained, similar to what was observed for gentamicin. In HEK293 cells transfected with six PTC-containing inserts, the tested compounds stimulated PTC-readthrough but with lower efficiency than aminoglycosides. The study allowed us to select compounds with minimal negative impact on cell viability and function but still the potential to induce PTC-readthrough.

Methods for the assessment of human airway ciliary function

“…State-of-the-art ciliary function analysis of airway epithelium underpins PCD diagnostics but also enables understanding of how cilia move in health or when temporarily damaged. Ciliary function analysis can underpin investigations of epithelial cell differentiation, integrity, disease, infection and drug therapy evaluation in airway culture models [41,[66][67][68].…”

“…If the effect of drug treatment on ciliary function is being assessed, it is important to consider pre-treatment baseline and temporal variability of ciliary function with drug action and half-life. Time-lapse coupled HSVA can facilitate continuous temporal cilia analysis of multiple experimental conditions in different wells, from specific x, y, z locations offering data repeatability [40,41]. The caveat of this method is that it relies on the ciliated cells remaining in situ e.g., nasal brushing samples grown on plastic or ALI-cultures on membranes, rather than free floating spheroids able to move out of position.…”

Ataluren—Promising Therapeutic Premature Termination Codon Readthrough Frontrunner

“…Ataluren was also tested as a potential readthrough therapeutic agent that could be used to treat primary ciliary dyskinesia (PCD) [ 104 ]. PCD symptoms include, among others, recurrent and chronic upper and lower airway infections.…”

“…What must be emphasized is that the observed stimulation potentials detected for gentamicin and G418 in this study were also lower as compared to previously determined. The differences observed may be caused by distortions of the fluc/rluc assay [ 104 ].…”

Molecular Approaches Fighting Nonsense

“…Additionally, for primary ciliary dyskinesia (PCD) caused by nonsense mutations, non-aminoglycosides readthrough therapies are an attractive option. A group of chemical compounds with known PTC-readthrough potential (ataluren, azithromycin, tylosin, amlexanox, and the experimental compound TC007) have been investigated, and the tested compounds stimulated PTC-readthrough with lower efficiency than aminoglycosides but had a minimal negative impact on cell viability and function [ 8 ]. Importantly, the treatment of inherited bone marrow failure syndromes (IBMFS) due to nonsense mutations in the respective IBMFS-related gene caused by the new generation of nonsense suppressor molecules and their mechanistic roles have been reviewed, with strengths and limitations emerging from preclinical and clinical studies [ 9 ].…”