Suppression of premature termination codons as a therapeutic approach

Abstract

In this review, we describe our current understanding of translation termination and pharmacological agents that influence the accuracy of this process. A number of drugs have been identified that induce suppression of translation termination at in-frame premature termination codons (PTCs; also known as nonsense mutations) in mammalian cells. We discuss efforts to utilize these drugs to suppress disease-causing PTCs that result in the loss of protein expression and function. In-frame PTCs represent a genotypic subset of mutations that make up ~11% of all known mutations that cause genetic diseases, and millions of patients have diseases attributable to PTCs. Current approaches aimed at reducing the efficiency of translation termination at PTCs (referred to as PTC suppression therapy) have the goal of alleviating the phenotypic consequences of a wide range of genetic diseases. Suppression therapy is currently in clinical trials for treatment of several genetic diseases caused by PTCs, and preliminary results suggest that some patients have shown clinical improvements. While current progress is promising, we discuss various approaches that may further enhance the efficiency of this novel therapeutic approach.

Figure 1

Model of eukaryotic translation termination. A complex comprised of eRF1 and eRF3 mediate translation termination. eRF1 recognizes any of the three stop codons (UAA, UAG, UGA) in the ribosomal A site. GTP hydrolysis by eRF3 assists: 1) stop codon recognition by eRF1, and 2) eRF1 accommodation into the peptidyl transferase center so polypeptide release can occur. [Note: a color version of this figure is available online.]

Figure 2

Model of PTC suppression. A) The eRF1/eRF3 complex efficiently mediates translation termination in eukaryotes, where incorporation of an amino acid carried by a near-cognate aminoacyl-tRNA into the nascent polypeptide is rare. B) Drugs such as aminoglycosides bind to the small ribosomal subunit to stimulate misreading at stop codons, leading to an increased frequency of near-cognate aminoacyl-tRNA incorporation that allows continued translation elongation in the correct ribosomal reading frame. [Note: a color version of this figure is available online.]

Figure 3

Structural changes that occur in the ribosomal decoding site. During proofreading of the codon-anticodon interaction, the bases of A1492 and A1493 of the 16S rRNA flip out and interact with the minor groove of the codon-anticodon helix. Shown are partial RNA structures of decoding sites from the 30S ribosomal subunit from Thermus thermophilus. These structures were obtained from 30S subunits in the (A) absence or (B) presence of paromomycin (shown in black). Structures were obtained using protein data bank numbers 1J5E (A) and 1IBK (B). [Note: a color version of this figure is available online.]

Figure 4

Key differences between the prokaryotic and eukaryotic decoding sites. E. coli numbering is shown for prokaryotes (orange), while S. cerevisiae numbering is shown for eukaryotes (green). Arrows indicate nucleotides that are key in decoding correct base pair interactions within the A site. The boxed nucleotides indicate differing residues between prokaryotic and eukaryotic decoding sites that affect the affinity of aminoglycoside binding. [Note: a color version of this figure is available online.]

Figure 5

Structures of non-aminoglycosides that induce PTC suppression in mammalian cells.

Figure 6

Structures of some aminoglycosides that induce PTC suppression in mammalian cells. A) Gentamicin isomers. B) Designer aminoglycoside derivatives. The boxed structures represent structural elements within conventional aminoglycosides that were used to generate the designer aminoglycosides NB30, NB54, and NB84. [Note: a color version of this figure is available online.]

Figure 7

Classes of mutations that cause cystic fibrosis. CFTR mutations are categorized into five classes (I-V) depending on the effect of the mutation on CFTR protein abundance, processing, localization, and function. The mutation classes are as follows: Class I = no CFTR protein is produced (includes nonsense mutations); Class II = CFTR processing is defective, so CFTR is retained in the ER and subsequently degraded; Class III = CFTR localizes to the cell surface, but is nonfunctional; Class IV = CFTR localizes to the cell surface, but with reduced activity; Class V = CFTR localizes to the cell surface and is functional, but its expression is reduced. [Note: a color version of this figure is available online.]

Figure 8

Rationale of combining suppression therapy with NMD inhibition. The presence of a nonsense mutation within an mRNA leads to premature translation termination, resulting in a truncated polypeptide that is nonfunctional and/or unstable. However, nonsense mutations often also trigger nonsense-mediated mRNA decay of the transcript that severely reduces its steady-state levels. The combination of these PTC-induced events contributes to the near complete loss of protein expression that often results in a disease state. Suppression therapy targets premature translation termination by inducing readthrough at PTCs. NMD inhibition could increase the pool of PTC-containing mRNAs available for translation and subsequent PTC suppression. The combination of suppression therapy and NMD inhibition simultaneously targets both PTC-mediated events to enhance protein restoration. [Note: a color version of this figure is available online.]

Figure 9

NMD inhibitors. UPF1 is a critical NMD factor that undergoes a cycle of phosphorylation and de-phosphorylation, which represents a pharmacological target for NMD inhibitors. SMG1, the kinase that phosphorylates UPF1, is inhibited by caffeine. The interaction between UPF1 and SMG5, which recruits PP2A phosphatase to dephosphorylate UPF1, is blocked by NMDI-1. [Note: a color version of this figure is available online.]