关键词: ataluren high-throughput screening (HTS) premature termination codon readthrough termination translation readthrough-inducing drug (TRID)

Mesh : Humans Codon, Nonsense Codon, Terminator Protein Biosynthesis Mutation Peptides / metabolism

来  源:   DOI:10.3390/biom13020242   PDF(Pubmed)

Abstract:
Premature termination codons (PTCs) account for ~12% of all human disease mutations. Translation readthrough-inducing drugs (TRIDs) are prominent among the several therapeutic approaches being used to overcome PTCs. Ataluren is the only TRID that has been approved for treating patients suffering from a PTC disease, Duchenne muscular dystrophy, but it gives variable readthrough results in cells isolated from patients suffering from other PTC diseases. We recently elucidated ataluren\'s mechanism of action as a competitive inhibitor of release factor complex (RFC) catalysis of premature termination and identified ataluren\'s binding sites on the ribosome responsible for such an inhibition. These results suggest the possibility of discovering new TRIDs, which would retain ataluren\'s low toxicity while displaying greater potency and generality in stimulating readthrough via the inhibition of termination. Here we present a detailed description of a new in vitro plate reader assay that we are using both to screen small compound libraries for the inhibition of RFC-dependent peptide release and to better understand the influence of termination codon identity and sequence context on RFC activity.
摘要:
过早终止密码子(PTC)占所有人类疾病突变的约12%。翻译连读诱导药物(TRID)在用于克服PTC的几种治疗方法中是突出的。Ataluren是唯一被批准用于治疗患有PTC疾病的患者的TRID,杜氏肌营养不良症,但是它在从患有其他PTC疾病的患者中分离出的细胞中给出了可变的读取结果。我们最近阐明了ataluren作为释放因子复合物(RFC)催化过早终止的竞争性抑制剂的作用机制,并确定了核糖体上负责这种抑制的ataluren结合位点。这些结果表明发现新的TRID的可能性,这将保留ataluren的低毒性,同时显示更大的效力和一般性,通过抑制终止刺激连读。在这里,我们提供了一种新的体外读板器测定法的详细描述,我们正在使用它来筛选小化合物文库以抑制RFC依赖性肽的释放,并更好地理解终止密码子同一性和序列上下文对RFC活性的影响。
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