2,6-Diaminopurine as a highly potent corrector of UGA nonsense mutations

Abstract

Nonsense mutations cause about 10% of genetic disease cases, and no treatments are available. Nonsense mutations can be corrected by molecules with nonsense mutation readthrough activity. An extract of the mushroom Lepista inversa has recently shown highefficiency correction of UGA and UAA nonsense mutations. One active constituent of this extract is 2,6-diaminopurine (DAP). In Calu-6 cancer cells, in which TP53 gene has a UGA nonsense mutation, DAP treatment increases p53 level. It also decreases the growth of tumors arising from Calu-6 cells injected into immunodeficient nude mice. DAP acts by interfering with the activity of a tRNA-specific 2′-O-methyltransferase (FTSJ1) responsible for cytosine 34 modification in tRNA Trp. Low-toxicity and high-efficiency UGA nonsense mutation correction make DAP a good candidate for the development of treatments for genetic diseases caused by nonsense mutations.

Suppression of Nonsense Mutations by New Emerging Technologies

“…Recently, it was reported that clitocine and 2,6-diaminopurine, found in extracts from the mushroom Lepista inversa , have a significant nonsense suppression activity [ 61 ]. More specifically, 2,6-diaminopurine could allow UGA PTCs to be read as a tryptophan codon [ 62 ]. As with all small-molecule nonsense suppressors, it needs to be determined whether the amino acid incorporated at the PTC will render a full-length functional protein.…”

Deciphering the molecular mechanism of stop codon readthrough

“…While PTCs do promote translation termination, they are more sensitive to readthrough than physiological stop codons. Although several studies using readthrough molecules have shown the absence of readthrough at physiological stop codons (Welch et al ., 2007; Benhabiles et al ., 2017; Trzaska et al ., 2020), it remains necessary to demonstrate for each new molecule that it does not impact translation termination at physiological stop codons.…”

“…It seems, however, that this is not an absolute rule, as some readthrough activators with no NMD‐inhibiting action promote greater synthesis of full‐length proteins than do dual‐action molecules. For instance, the readthrough activators Lepista flaccida extract H7 and 2,6‐diaminopurine, which do not inhibit NMD, correct UGA and UAA (extract H7) or UGA only (2,6‐diaminopurine) more effectively than dual‐action G418 (Correa‐Cerro et al ., 2005; Benhabiles et al ., 2017; Trzaska et al ., 2020).…”

“…Clitocine is another molecule with a high readthrough‐promoting capacity. However, this molecule, found in various mushroom species (including Leucopaxillus giganteus and Lepista flaccida ) (Kubo et al ., 1986; Fortin et al ., 2006; Wilde et al ., 2007; Ren et al ., 2008; Benhabiles et al ., 2017; Trzaska et al ., 2020) unfortunately shows high toxicity, which limits its potential therapeutic development (Fortin et al ., 2006; Sun et al ., 2012). The mode of action of clitocine has been studied.…”

“…The luciferase activity measured in this screening system thus depends on the efficiency of readthrough occurring on the number of firefly luciferase mRNAs that have escaped NMD. This screening system has been used successfully to identify Lepista flaccida fungus extract H7 and 2,6‐diaminopurine as two highly potent correctors of nonsense mutations in human cells (Benhabiles et al ., 2017; Trzaska et al ., 2020).…”