Identifying Potent Nonsense-Mediated mRNA Decay Inhibitors with a Novel Screening System.

Nonsense-mediated mRNA decay (NMD) is a quality control mechanism that degrades mRNAs carrying a premature termination codon. Its inhibition, alone or in combination with other approaches, could be exploited to develop therapies for genetic diseases caused by a nonsense mutation. This, however, requires molecules capable of inhibiting NMD effectively without inducing toxicity. We have built a new screening system and used it to identify and validate two new molecules that can inhibit NMD at least as effectively as cycloheximide, a reference NMD inhibitor molecule. These new NMD inhibitors show no cellular toxicity at tested concentrations and have a working concentration between 6.2 and 12.5 µM. We have further validated this NMD-inhibiting property in a physiopathological model of lung cancer in which the TP53 gene carries a nonsense mutation. These new molecules may potentially be of interest in the development of therapies for genetic diseases caused by a nonsense mutation.

Use of 2,6-diaminopurine as a potent suppressor of UGA premature stop codons in cystic fibrosis.

Nonsense mutations are responsible for around 10% of cases of genetic diseases, including cystic fibrosis. 2,6-diaminopurine (DAP) has recently been shown to promote efficient readthrough of UGA premature stop codons. In this study, we show that DAP can correct a nonsense mutation in the Cftr gene in vivo in a new CF mouse model, in utero, and through breastfeeding, thanks, notably, to adequate pharmacokinetic properties. DAP turns out to be very stable in plasma and is distributed throughout the body. The ability of DAP to correct various endogenous UGA nonsense mutations in the CFTR gene and to restore its function in mice, in organoids derived from murine or patient cells, and in cells from patients with cystic fibrosis reveals the potential of such readthrough-stimulating molecules in developing a therapeutic approach. The fact that correction by DAP of certain nonsense mutations reaches a clinically relevant level, as judged from previous studies, makes the use of this compound all the more attractive.

A role for AKT1 in nonsense-mediated mRNA decay.

Nonsense-mediated mRNA decay (NMD) is a highly regulated quality control mechanism through which mRNAs harboring a premature termination codon are degraded. It is also a regulatory pathway for some genes. This mechanism is subject to various levels of regulation, including phosphorylation. To date only one kinase, SMG1, has been described to participate in NMD, by targeting the central NMD factor UPF1. Here, screening of a kinase inhibitor library revealed as putative NMD inhibitors several molecules targeting the protein kinase AKT1. We present evidence demonstrating that AKT1, a central player in the PI3K/AKT/mTOR signaling pathway, plays an essential role in NMD, being recruited by the UPF3X protein to phosphorylate UPF1. As AKT1 is often overactivated in cancer cells and as this should result in increased NMD efficiency, the possibility that this increase might affect cancer processes and be targeted in cancer therapy is discussed.