Identification and Characterization of Small Molecules That Inhibit Nonsense-Mediated RNA Decay and Suppress Nonsense p53 Mutations

Abstract

Many of the gene mutations found in genetic disorders, including cancer, result in premature termination codons (PTCs) and the rapid degradation of their mRNAs by nonsense mediated RNA decay (NMD). We used virtual library screening (VLS) targeting a pocket in the SMG7 protein, a key component of the NMD mechanism, to identify compounds that disrupt the SMG7-UPF1 complex and inhibit NMD. Several of these compounds upregulated NMD targeted mRNAs at nanomolar concentrations with minimal toxicity in cell based assays. As expected, pharmacological NMD inhibition disrupted SMG7-UPF1 interactions. When used in cells with PTC mutated p53, pharmacological NMD inhibition combined with a PTC “read-through” drug led to restoration of full-length p53 protein, upregulation of p53 downstream transcripts, and cell death. These studies serve as proof-of-concept that pharmacological NMD inhibitors can restore mRNA integrity in the presence of PTC and be used as part of a strategy to restore full length protein in a variety of genetic diseases.

Caspases shutdown nonsense-mediated mRNA decay during apoptosis

“…38 Other examples include NMD inhibition by amlexanox where there is no effect on cell viability, 29 or more recently, by a new set of NMD inhibitors targeting the interaction between UPF1 and SMG7 proteins with a low cellular toxicity. 39 Whether NMD inhibition can activate apoptosis might be dependent on the degree of NMD inhibition.…”

Nonsense-mediated mRNA decay in humans at a glance

“…In contrast to Ataluren, cardiac glycosides, which elevate the level of intracellular Ca 2+ , are capable of inhibiting NMD at concentrations that reportedly do not affect cellular viability (Nickless et al, 2014). It might be possible to concomitantly use Ataluren and cardiac glycosides, or possibly a clinically effective nonsense suppressor with either another small reagent that has been reported to inhibit NMD (Bhuvanagiri et al, 2014;Martin et al, 2014;Dang et al, 2009;Feng et al, 2015;Gopalsamy et al, 2012;Usuki et al, 2004) or one or more antisense oligonucleotides that occlude deposition of the one or more EJCs that would normally reside downstream of a particular PTC (Nomakuchi et al, in press). Another approach that might be worthwhile for obtaining full-length protein from diseaseassociated mRNAs is site-directed pseudouridylation of in-frame PTCs (Karijolich and Yu, 2011); by providing a gene-specific therapeutic strategy, such a strategy would be expected to have only minimal toxic side effects.…”

Leveraging Rules of Nonsense-Mediated mRNA Decay for Genome Engineering and Personalized Medicine

“…Small molecules inhibiting the core NMD machinery may offer a useful additional treatment, and efforts to discover such molecules are ongoing (Martin et al, 2014). Given the roles that NMD plays beyond quality control, these molecules may also be useful in other situations.…”