Mechanism-based approach in designing patient-specific combination therapies for nonsense mutation diseases

Premature termination codon (PTC) diseases, arising as a consequence of nonsense mutations in a patient's DNA, account for approximately 12% of all human disease mutations. Currently there are no FDA approved treatments for increasing PTC readthrough in nonsense mutation diseases, although one translational readthrough inducing drug, ataluren, has had conditional approval for treatment of Duchenne muscular dystrophy in Europe and elsewhere for 10 years. Ataluren displays consistent low toxicity in clinical trials for treatment of several different PTC diseases, but its therapeutic effects on such diseases are inconsistent. The identity of the stop codon and its sequence context are major determinants of PTC readthrough efficiency in both the absence and presence of nonsense suppressors. Previously we have shown that ataluren stimulates readthrough exclusively by competitively inhibiting release factor complex (RFC, eRF1.eRF3.GTP)-dependent catalysis of translation termination. Here, using an in vitro reconstituted system (PURE-LITE) and both ensemble and single molecule assays, we demonstrate that PTC identity and the immediately adjacent mRNA sequence contexts modulate the catalytic activity of RFC in terminating peptide elongation. Such modulation largely determines the effectiveness of ataluren in stimulating readthrough, whether added alone or in combination with either the aminoglycoside G418 or an anticodon edited aa-tRNA, each of which stimulate readthrough by mechanisms orthogonal to that of ataluren. Our results provide an attractive rationale for the variability of ataluren effectiveness in stimulating readthrough in clinical trials. Patients harboring a PTC mutation with a sequence context promoting strong interaction with RFC are predicted to be resistant to ataluren, whereas ataluren treatment should be more effective for patient sequences conferring weaker interaction with RFC.