Characterization of new-generation aminoglycoside promoting premature termination codon readthrough in cancer cells

Abstract

Nonsense mutations, generating premature termination codons (PTCs), account for 10% to 30% of the mutations in tumor suppressor genes. Nonsense translational suppression, induced by small molecules including gentamicin and G418, has been suggested as a potential therapy to counteract the deleterious effects of nonsense mutations in several genetic diseases and cancers. We describe here that NB124, a synthetic aminoglycoside derivative recently developed especially for PTC suppression, strongly induces apoptosis in human tumor cells by promoting high level of PTC readthrough. Using a reporter system, we showed that NB124 suppressed several of the PTCs encountered in tumor suppressor genes, such as the p53 and APC genes. We also showed that NB124 counteracted p53 mRNA degradation by nonsense-mediated decay (NMD). Both PTC suppression and mRNA stabilization contributed to the production of a full-length p53 protein capable of activating p53-dependent genes, thereby specifically promoting high levels of apoptosis. This new-generation aminoglycoside thus outperforms the only clinically available readthrough inducer (gentamicin). These results have important implications for the development of personalised treatments of PTC-dependent diseases and for the development of new drugs modifying translation fidelity.

A new in vitro assay measuring direct interaction of nonsense suppressors with the eukaryotic protein synthesis machinery

“…The saturation curves obtained with the 7 aminoglycosides (AGs) examined ( Figure 3A) are each consistent with a single tight site of AG binding to the ribosome which induces readthrough, with EC 50 s falling in the range of 0.4 -4 µM and plateau octapeptide/POST5 ratios varying from 0.1 -0.3 (Table S1) above the basal level measured in the absence of added NonSup (see Supporting Information, Item 2). These results are consistent with results on readthrough obtained in intact cells showing that a) G418, NB84, NB124 23 and gentamicin X2 24 are much more effective than the gentamicin mixture currently approved as an antibiotic; and b) NB84, NB124, 23 and gentamicin X2 24 have similar potencies, measured by either EC 50 or readthrough efficiency. These consistencies suggest that aminoglycosides stimulate readthrough in cells primarily by direct effects on the protein synthesis machinery.…”

“…EC 50 values found in intact cells differ considerably from those measured by our cosedimentation assay, being much higher for AGs, 23,30 and much lower for ataluren, 8,31 RTC13 25 and GJ072. 25 We attribute these differences to the poor uptake of positively charged aminoglycosides into cells, while uptake is favored for the hydrophobic ataluren-like molecules.…”

Resorting the function of the colorectal cancer gatekeeper adenomatous polyposis coli

“…There have been a number of previous clinical and translational studies that examined the applicability of the read‐through approach to a clinical setting . Though some of these studies focused on the cancer arena, none resulted in clinical trials . Therefore, the ability to induce read‐through of the APC full‐length protein by antibiotic treatment represents a new potential strategy to delay or even prevent cancer formation in FAP patients.…”

“…29 Though some of these studies focused on the cancer arena, none resulted in clinical trials. [30][31][32] Therefore, the ability to induce read-through of the APC fulllength protein by antibiotic treatment represents a new potential strategy to delay or even prevent cancer formation in FAP patients. Indeed, reports by our group have already demonstrated the efficacy of aminoglycosides and macrolide induced readthrough in a multiple intestinal neoplasm APC (Min/+) mouse model, 21 and of other macrolide compounds in vitro.…”

Screening Readthrough Compounds to Suppress Nonsense Mutations: Possible Application to β-Thalassemia

“…NB124 was found able to restore the full length synthesis of CFTR and chloride transport in an animal genetic model and rescue about 7% of CFTR function in primary human bronchial epithelial (HBE) CF cells [59]. Most recently, NB124 was identified as a potent nonsense suppressor of several nonsense mutations located in the p53 and APC (adenomatous polyposis coli) tumor suppressor genes, which account for 10% and 30% of mutations in human cancers, respectively [60]. NB124 restores the full length expression of p53, which is functional in inducing the transcription of its target genes.…”