Synthetic Aminoglycosides Efficiently Suppress Cystic Fibrosis Transmembrane Conductance Regulator Nonsense Mutations and Are Enhanced by Ivacaftor

Xue, Xiaojiao; Mutyam, Venkateshwar; Tang, Liping; Biswas, Sanket; Du, Ming; Jackson, Laura A.; Dai, Yuling; Belakhov, V. V.; Shalev, Moran; Chen, Fuquan; Schacht, Jochen; Bridges, Robert J.; Baasov, Timor; Hong, Jeong S.; Bedwell, David M.; Rowe, Steven M.

doi:10.1165/rcmb.2013-0282oc

Cited by 138 publications

(137 citation statements)

References 54 publications

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“…Although our studies have addressed substitutions in only a limited number of positions in luciferase, His3, and CFTR, it is nevertheless reassuring with respect to the potential of therapeutic nonsense suppression that all of the substitutions analyzed here retained some degree of function. It will be of interest to determine whether the observed activities can be increased by parallel approaches, e.g., antagonists of mRNA decay (2), specific protein correctors or potentiators [as have been successfully used for CFTR (39)], or enhancers of the expression of certain tRNAs.…”

Section: Discussionmentioning

confidence: 99%

Ataluren stimulates ribosomal selection of near-cognate tRNAs to promote nonsense suppression

Roy

Friesen

Tomizawa

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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189

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A premature termination codon (PTC) in the ORF of an mRNA generally leads to production of a truncated polypeptide, accelerated degradation of the mRNA, and depression of overall mRNA expression. Accordingly, nonsense mutations cause some of the most severe forms of inherited disorders. The small-molecule drug ataluren promotes therapeutic nonsense suppression and has been thought to mediate the insertion of near-cognate tRNAs at PTCs. However, direct evidence for this activity has been lacking. Here, we expressed multiple nonsense mutation reporters in human cells and yeast and identified the amino acids inserted when a PTC occupies the ribosomal A site in control, ataluren-treated, and aminoglycoside-treated cells. We find that ataluren’s likely target is the ribosome and that it produces full-length protein by promoting insertion of near-cognate tRNAs at the site of the nonsense codon without apparent effects on transcription, mRNA processing, mRNA stability, or protein stability. The resulting readthrough proteins retain function and contain amino acid replacements similar to those derived from endogenous readthrough, namely Gln, Lys, or Tyr at UAA or UAG PTCs and Trp, Arg, or Cys at UGA PTCs. These insertion biases arise primarily from mRNA:tRNA mispairing at codon positions 1 and 3 and reflect, in part, the preferred use of certain nonstandard base pairs, e.g., U-G. Ataluren’s retention of similar specificity of near-cognate tRNA insertion as occurs endogenously has important implications for its general use in therapeutic nonsense suppression.

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Section: Discussionmentioning

confidence: 99%

Ataluren stimulates ribosomal selection of near-cognate tRNAs to promote nonsense suppression

Roy

Friesen

Tomizawa

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

189

218

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“…Interestingly, compounds 10 and 12 were shown to have read-trough activity similar to G418 in both in-vitro and ex-vivo systems, but exhibited a significantly reduced cell toxicity. Furthermore, one of these lead structures, compound 9 (also named NB124) restored CFTR function to ~7% of wild type activity in primary human bronchial epithelial (HBE) CF cells (G542X/delF508), a highly relevant preclinical model with endogenous CFTR expression, and rescued CFTR function in a CF mouse model expressing a human CFTR-G542X transgene; efficacy was superior to gentamicin and exhibited favorable pharmacokinetic properties, suggesting in vitro results translated to clinical benefit in vivo 83 .…”

Section: Challenges In the Design Of Novel Nonsense Read-through Imentioning

confidence: 99%

When proteins start to make sense: fine-tuning of aminoglycosides for PTC suppression therapy

Shalev

Baasov

2014

Med. Chem. Commun.

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Aminoglycosides (AGs) are highly potent antibacterial agents, which are known to exert their deleterious effects on bacterial cells by interfering with the translation process, leading to aberrant protein synthesis that usually results in cell death. Nearly 45 years ago, AGs were shown to induce read-through activity in prokaryotic systems by selectively encoding tRNA molecules at premature termination codon (PTC) positions; resulting in the generation of full length functional proteins. However, only in the last 20 years this ability has been demonstrated in eukaryotic systems, highlighting their potential as therapeutic agents to treat PTC induced genetic disorders. Despite the great potential, AGs use in these manners is quite restricted due to relatively high toxicity values observed upon their administration. Over the last few years several synthetic derivatives were developed to overcome some of the enhanced toxicity issues, while in parallel showed significantly improved PTC suppression activity in various in-vitro, ex-vivo and in-vivo models of a variety of different diseases models underling by PTC mutations. Although these derivatives hold great promise to serve as therapeutic candidates they also demonstrate the necessity to further understand the molecular mechanisms of which AGs confer their biological activity in eukaryotic cells for further rational drug design. Recent achievements in structural research shed light on AGs mechanism of action and opened a new avenue in the development of new and improved therapeutic derivatives. The following manuscript highlights these accomplishments and summarizes their contributions to the state of art rational drug design.

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“…Transcript encoding W1282X-CFTR was seen in nasal epithelial cells of homozygous W1282X CF subjects with 30 -80% abundance compared with that of wild type CFTR in non-CF subjects (13). In bronchial epithelial cells virally infected to express W1282X-CFTR cDNA, butyrate-induced up-regulation of truncated CFTR expression produced cAMP/genistein-stimulated chloride conductance (14), and in the same cell model, VX-770 produced an ϳ4-fold increase in CFTR activity despite the apparent absence of full-length protein (15). Patch clamp experiments showed that VX-770 in combination with curcumin produced significant activation of W1282X-CFTR despite evidence of impaired ATP-dependent NBD1-NBD2 dimerization (16).…”

mentioning

confidence: 98%

Correctors and Potentiators Rescue Function of the Truncated W1282X-Cystic Fibrosis Transmembrane Regulator (CFTR) Translation Product

Haggie

Phuan

Tan

et al. 2017

Journal of Biological Chemistry

111

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Edited by Thomas Sö llnerW1282X is the fifth most common cystic fibrosis transmembrane regulator (CFTR) mutation that causes cystic fibrosis. Here, we investigated the utility of a small molecule corrector/ potentiator strategy, as used for ⌬F508-CFTR, to produce functional rescue of the truncated translation product of the W1282X mutation, CFTR 1281 , without the need for readthrough. In transfected cell systems, certain potentiators and correctors, including VX-809 and VX-770, increased CFTR 1281 activity. To identify novel correctors and potentiators with potentially greater efficacy on CFTR 1281 , functional screens were done of ϳ30,000 synthetic small molecules and drugs/nutraceuticals in CFTR 1281 -transfected cells. Corrector scaffolds of 1-arylpyrazole-4-arylsulfonyl-piperazine and spiro-piperidine-quinazolinone classes were identified with up to ϳ5-fold greater efficacy than VX-809, some of which were selective for CFTR 1281 , whereas others also corrected ⌬F508-CFTR. Several novel potentiator scaffolds were identified with efficacy comparable with VX-770; remarkably, a phenylsulfonamide-pyrrolopyridine acted synergistically with VX-770 to increase CFTR 1281 function ϳ8-fold over that of VX-770 alone, normalizing CFTR 1281 channel activity to that of wild type CFTR. Corrector and potentiator combinations were tested in primary cultures and conditionally reprogrammed cells generated from nasal brushings from one W1282X homozygous subject. Although robust chloride conductance was seen with correctors and potentiators in homozygous ⌬F508 cells, increased chloride conductance was not found in W1282X cells despite the presence of adequate transcript levels. Notwithstanding the negative data in W1282X cells from one human subject, we speculate that corrector and potentiator combinations may have therapeutic efficacy in cystic fibrosis caused by the W1282X mutation, although additional studies are needed on human cells from W1282X subjects.

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Synthetic Aminoglycosides Efficiently Suppress Cystic Fibrosis Transmembrane Conductance Regulator Nonsense Mutations and Are Enhanced by Ivacaftor

Cited by 138 publications

References 54 publications

Ataluren stimulates ribosomal selection of near-cognate tRNAs to promote nonsense suppression

Ataluren stimulates ribosomal selection of near-cognate tRNAs to promote nonsense suppression

When proteins start to make sense: fine-tuning of aminoglycosides for PTC suppression therapy

Correctors and Potentiators Rescue Function of the Truncated W1282X-Cystic Fibrosis Transmembrane Regulator (CFTR) Translation Product

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