The nucleoside analog clitocine is a potent and efficacious readthrough agent

Abstract

Nonsense mutations resulting in a premature stop codon in an open reading frame occur in critical tumor suppressor genes in a large number of the most common forms of cancers and are known to cause or contribute to the progression of disease. Low molecular weight compounds that induce readthrough of nonsense mutations offer a new means of treating patients with genetic disorders or cancers resulting from nonsense mutations. We have identified the nucleoside analog clitocine as a potent and efficacious suppressor of nonsense mutations. We determined that incorporation of clitocine into RNA during transcription is a prerequisite for its readthrough activity; the presence of clitocine in the third position of a premature stop codon directly induces readthrough. We demonstrate that clitocine can induce the production of p53 protein in cells harboring p53 nonsense-mutated alleles. In these cells, clitocine restored production of full-length and functional p53 as evidenced by induced transcriptional activation of downstream p53 target genes, progression of cells into apoptosis, and impeded growth of nonsense-containing human ovarian cancer tumors in xenograft tumor models. Thus, clitocine induces readthrough of nonsense mutations by a previously undescribed mechanism and represents a novel therapeutic modality to treat cancers and genetic diseases caused by nonsense mutations.

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

“…For 5 weeks, the mice were forced-fed every day with DMSO or DAP and tumor growth was monitored three times weekly. We assumed that if DAP promotes synthesis of a functional p53 protein in Calu-6 cells, these cells should divide more slowly and enter apoptosis as previously reported 24 . After 5 weeks of treatment, a significant difference was observed between Calu-6cell-injected mice exposed to DMSO and ones exposed to DAP (Fig.…”

“…1c). The combined use of nuclear magnetic resonance (NMR) and mass spectrometry allowed identifying the major component in fractions F13 and F15 as clitocine ((2R,3R,4S,5R)-2-[(6-amino-5nitropyrimidin-4-yl)amino]-5-(hydroxymethyl)oxolane-3,4-diol) (76% and 47%, respectively), known as a potent readthrough molecule 24 . In fraction F10 another molecule, showing exclusive readthrough of the UGA stop codon and present at about 77%, was assigned as DAP on the basis of the 1 H and 13 C NMR and mass fragmentation data (Supplementary Table 1, Supplementary Figs.…”

“…We have identified two molecules responsible for the activity of H7, one being clitocine, previously shown to correct nonsense mutations more efficiently than G418 (ref. 24 ). The mode of action of clitocine differs from that of other readthrough activator molecules, such as aminoglycosides, shown to interact with the ribosome 50 .…”

“…Several molecules have been shown to activate readthrough, including aminoglycosides, such as gentamicin and G418 and non-aminoglycosides, such as PTC124/ataluren/Translarna and RTC13/14 (refs. [21][22][23][24][25][26][27][28][29] ). Some of the compounds used, such as G418 and amlexanox, can both inhibit NMD and activate readthrough 19,30 .…”

Succinic Semialdehyde Dehydrogenase Deficiency: An Update

“…In cell culture, amlexanox was able to rescue the expression of functional proteins from mRNAs containing nonsense mutations for p53, dystrophin, aspartylglucosaminidase, and type VII collagen [127,157,159]. Further potentially efficacious read-through agents include the nucleoside analog clitocine and components of fungi extracts that were identified in a recent drug screen [160,161]. Since the efficacy of these drugs is also dependent on their capability to cross the BBB, further research will be required to test if these drugs might also provide a therapy option for SSADH-D. Interestingly, the findings of Akaboshi and colleagues suggested that nonsense variants, especially Trp204X and Arg412X substitutions, are frequently present in SSADH-D [31].…”