TY - JOUR T1 - ELX-02 generates protein via premature stop codon read-through without inducing native stop codon read-through proteins JF - Journal of Pharmacology and Experimental Therapeutics JO - J Pharmacol Exp Ther DO - 10.1124/jpet.120.265595 SP - jpet.120.265595 AU - Daniel Crawford AU - Iris Alroy AU - Neal Sharpe AU - Matthew Goddeeris AU - Greg Williams Y1 - 2020/01/01 UR - http://jpet.aspetjournals.org/content/early/2020/05/06/jpet.120.265595.abstract N2 - ELX-02 is a clinical stage, small molecule Eukaryotic Ribosomal Selective Glycoside (ERSG) acting to induce read-through of premature stop codons (PSCs) that results in translation of full-length protein. However, improved read-through at PSCs has raised the question of whether native stop codon (NSC) fidelity would be impacted. Here, we compare read-through by ELX-02 in PSC and NSC contexts. DMS-114 cells containing a PSC in the TP53 gene were treated with ELX-02 and tested for increased nuclear p53 protein expression, while also monitoring two other proteins for NSC read-through. Additionally, blood samples were taken from healthy subjects pre- and post-treatment with ELX-02 (0.3-7.5mg/kg). These samples were processed to collect white blood cells, then analyzed by western blot to identify native and potentially elongated proteins from NSC read-through. In a separate experiment, lymphocytes cultivated with vehicle or ELX-02 (20 and 100μg/mL) were subjected to proteomic analysis. We found that ELX-02 produced significant read-through of the PSC found in TP53 mRNA in DMS-114 cells, resulting in increased p53 protein expression and consistent with decreased nonsense mediated mRNA degradation. NSC read-through protein products were not observed in either DMS-114 cells or in clinical samples from subjects dosed with ELX-02. The number of read-through proteins identified using proteomic analysis was lower than estimated and none of the NSC read-through products identified with >2 peptides showed dose-dependent responses to ELX-02. Our results demonstrate significant PSC read-through by ELX-02 with maintained NSC fidelity, thus supporting the therapeutic utility of ELX-02 in diseases resulting from nonsense alleles.SIGNIFICANCE STATEMENT ELX-02 produces significant read-through of premature stop codons leading to full-length functional protein, demonstrated here using the R213X mutation in the TP53 gene of DMS-114 cells. In addition, three complementary techniques suggest that ELX-02 does not promote read-through of native stop codons at concentrations that lead to premature stop codon read-through. Thus, ELX-02 may be a potential therapeutic option for nonsense mutation-mediated genetic diseases. ER -