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RTC 13 and RTC14

Large numbers of genetic disorders are caused by nonsense mutations for which compound-induced readthrough of premature termination codons (PTCs) might be exploited as a potential treatment strategy. We have successfully developed a sensitive and quantitative high-throughput screening (HTS) assay, protein transcription/translation (PTT)–enzyme-linked immunosorbent assay (ELISA), for identifying novel PTC-readthrough compounds using ataxia-telangiectasia (A-T) as a genetic disease model. This HTS PTT-ELISA assay is based on a coupled PTT that uses plasmid templates containing prototypic A-T mutated (ATM) mutations for HTS. The assay is luciferase independent. We screened ∼34,000 compounds and identified 12 low-molecular-mass nonaminoglycosides with potential PTC-readthrough activity. From these, two leading compounds consistently induced functional ATM protein in ATM-deficient cells containing disease-causing nonsense mutations, as demonstrated by direct measurement of ATM protein, restored ATM kinase activity, and colony survival assays for cellular radiosensitivity. The two compounds also demonstrated readthrough activity in mdx mouse myotube cells carrying a nonsense mutation and induced significant amounts of dystrophin protein.

Translation termination is signaled by three stop codons: UAA, UAG, and UGA. This mechanism is highly conserved, although each stop codon has a different efficiency for terminating translation. UGA is considered to be a “leaky” stop codon with the highest intrinsic readthrough potential. UAA shows high fidelity and little intrinsic readthrough potential, whereas UAG has intermediate fidelity (Weiner and Weber, 1973; Lovett et al., 1991). Nonsense mutations create primary premature termination codons (PTCs) and result in either no formation of the target protein or truncated protein with impaired stability.
Certain compounds influence the fidelity of stop codon recognition and induce readthrough of primary PTCs, which allows translation of some full-length protein. In many cases, the readthrough-induced protein is functional, even when it contains a wrongly incorporated amino acid (Keeling and Bedwell, 2005; Zingman et al., 2007).

It is estimated that 30% of human disease-causing alleles are nonsense mutations (Mendell and Dietz, 2001). Other types of mutation, such as frameshift and splicing mutations, lead to secondary PTCs; however, these are not therapeutic targets for readthrough compounds (RTCs). Considering that >1,800 distinct genetic disorders are caused by nonsense mutations, the readthrough of primary PTCs has treatment potential for large numbers of patients.

To date, most reported PTC-RTCs that are active in mammalian cells have belonged to the aminoglycoside antibiotics class (Keeling and Bedwell, 2005; Zingman et al., 2007). Certain types of aminoglycosides can induce ribosomes to read through PTC mutations via insertion of a random amino acid by near-cognate transfer RNA. The therapeutic potential of aminoglycosides has been evaluated in the laboratory for different genetic models, such as cystic fibrosis (Howard et al., 1996; Bedwell et al., 1997; Du et al., 2002), muscular dystrophy (Howard et al., 2000; Wagner et al., 2001; Dunant et al., 2003; Loufrani et al., 2004), Hurler syndrome (Keeling et al., 2001), cystinosis (Helip-Wooley et al., 2002), spinal muscular atrophy (Sossi et al., 2001), ataxia-telangiectasia (A-T; Lai et al., 2004), and type 1 Usher syndrome (Rebibo-Sabbah et al., 2007). Clinical trials also indicate that aminoglycosides can induce some functional protein production; however, the therapeutic benefits remain uncertain (Wilschanski et al., 2000; Clancy et al., 2001; Wagner et al., 2001; Politano et al., 2003). Furthermore, the toxicity of most commercial aminoglycosides in mammals has greatly diminished their potential for successful readthrough therapy (Mingeot-Leclercq and Tulkens, 1999; Guan et al., 2000). Therefore, efforts are underway to develop better aminoglycoside derivatives with reduced toxicity and enhanced activity (Nudelman et al., 2006; Rebibo-Sabbah et al., 2007). Recently, PTC Therapeutics (South Plainfield, NJ) described a more efficient nonaminoglycoside RTC, PTC124, which was developed synthetically by screening >800,000 chemicals and analogues using a luciferase-based high-throughput screening (HTS) assay (Welch et al., 2007; Hirawat et al., 2007; M. Du et al., 2008). A phase-I clinical study in cystic fibrosis confirmed that PTC124 is generally well tolerated and appears to have more efficient readthrough activity than aminoglycosides (Hirawat et al., 2007). Moreover, PTC124 does not induce ribosomal readthrough of normal stop codons. A phase-II clinical trial is underway (Kerem et al., 2008). However, a recent study indicates that the initial discovery of PTC124 by HTS may have been biased by its direct effect on the FLuc (firefly luciferase) reporter used (Auld et al., 2009), indicating the importance of a luciferase-independent HTS assay for future drug screening.
In an effort to discover new RTCs, we developed a sensitive and quantitative luciferase-independent HTS assay, protein transcription/translation (PTT)–ELISA. The PTT-ELISA assay was validated for a fully automated 384-well robotic platform and used to screen ∼34,000 compounds. We focused followup efforts on 12 low-molecular-mass nonaminoglycoside compounds. From there, we identified two compounds that induced low levels of full-length functional A-T mutated (ATM) protein in A-T cells carrying ATM nonsense mutations, as demonstrated by direct measurement of ATM protein using ATM-ELISA, ATM-Ser1981 autophosphorylation, trans-phosphorylation of structural maintenance of chromosome (SMC) 1–Ser966, and colony survival assay (CSA). Both compounds also showed PTC-readthrough activity in mdx mouse myotube cells carrying a nonsense mutation and induced significant amounts of dystrophin protein.

Collectively, these studies provide the first robust luciferase-independent HTS assay for identifying RTCs and proof of principle for PTC readthrough by nonaminoglycoside compounds. They further establish that enhanced PTC readthrough can be considered as a therapeutic strategy for correcting nonsense mutations in many genetic diseases.

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