Overcoming Nonsense Mutations by ...

DISCOVERY

nature publishing group

Aminoglycoside-induced Translational Read-through in Disease: Overcoming Nonsense Mutations by Pharmacogenetic Therapy LV Zingman1,2, S Park1,2, TM Olson1,2, AE Alekseev1,2 and A Terzic1,2 A third of inherited diseases result from premature termination codon mutations. Aminoglycosides have emerged as vanguard pharmacogenetic agents in treating human genetic disorders due to their unique ability to suppress gene translation termination induced by nonsense mutations. In preclinical and pilot clinical studies, this therapeutic approach shows promise in phenotype correction by promoting otherwise defective protein synthesis. The challenge ahead is to maximize efficacy while preventing interaction with normal protein production and function.

The information encoded in DNA and the proper decoding into proteins are fundamental to life.1 Change in the genetic code brings the danger of life-threatening disease. There are more than 1,800 inherited human diseases caused by nonsense mutations, i.e., alterations in the genetic code that prematurely stop the translation of proteins.2,3 Indeed, about 30 percent of heritable disorders result from premature termination codon (PTC) mutations.2,3 This frequency may be even higher in certain syndromes or in specific human populations. Moreover, different classes of mutations, including frameshift insertions and/or deletions, splice-site intron inclusions, or simple substitutions of a normal codon with UAA, UAG, or UGA codons, can all introduce a premature stop signal, thus arresting protein synthesis.4 Wellknown examples include Duchenne muscular dystrophy, the most common X-linked fatal genetic disease, where 410–20 percent of patients carry a nonsense mutation resulting in premature translation termination. Similarly, in cystic fibrosis, B2–5 percent of patients (60 percent for Ashkenazi Jews) have nonsense mutations in the cystic fibrosis transmembrane regulator gene.2,4–6 PTC mutations in

tumor-suppressor genes are also common in the development and progression of cancer.2 At the molecular level, PTC mutations have major consequences. Typically, a PTC mutation precipitates rapid degradation of the mRNA carrying the premature codon, after a single cycle of translation, through nonsense-mediated mRNA decay. Activation of this mechanism, while protective against potentially lethal production of truncated protein by nullifying deleterious suppression of synthesis and/or function of vital cell proteins, may not prevent the development of a disease phenotype due to the absence of the translationally defective protein itself.2,4,7 In the absence of nonsensemediated mRNA decay, truncated polypeptides could be produced.2,4,7 Assembly of a truncated protein in vivo is, however, a rare event, and is usually related to a PTC positioned within the last exon or in a splicing region resulting in mutation-induced exon skipping. The biological effect of a mutated gene may be difficult to predict, as it depends on the extent of protein truncation, stability of the polypeptide product, and degree of interference with the expression of the normal allele (e.g., dominant-negative effect).2,7 Two major therapeutic approaches to overcome nonsense mutations are presently considered. Gene therapy is, in principle, the treatment of choice due to the potential for targeted repair, but, to date, several limitations have precluded clinical success.3,7 Alternatively, pharmacological approaches can modify gene expression and block nonsensemediated mRNA destruction, without correcting the underlying gene defect. The rationale supporting such emerging pharmacogenetic strategies is the premise that even limited expression of a mutated gene could result in the production of a partially or fully functional protein, sufficient for therapeutic benefit.2,4,6,7

1

Marriott Heart Disease Research Program, Department of Medicine, Division of Cardiovascular Diseases, Mayo Clinic, Rochester, Minnesota, USA; 2Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, Minnesota, USA. Correspondence: LV Zingman ([email protected])

doi:10.1038/sj.clpt.6100012 CLINICAL PHARMACOLOGY & THERAPEUTICS | VOLUME 81 NUMBER 1 | JANUARY 2007

99

DISCOVERY

AMINOGLYCOSIDES: PROTEIN TRANSLATION-MODIFYING DRUGS

Aminoglycosides are widely used in clinical practice as bactericidal antibiotics with established effects on translational accuracy or efficiency.8 Their utility as antibacterial agents arises from binding to the highly conserved 16S ribosomal RNA (rRNA) at the decoding center (Figure 1). This center normally facilitates accurate codon–anticodon pairing. In the presence of an aminoglycoside, the conformation of rRNA becomes altered, inducing codon misreading that causes either incorporation of an erroneous amino acid (mis-incorporation) at a sense codon or failure of recognition of the stop codon, leading to translational read-through rather than chain termination (Figure 2).9–11 Although aminoglycosides target a conserved region of the rRNA sequence, these agents are highly active against bacterial and mitochondrial ribosomes, but have limited interaction with human ribosomes. The specificity is related to the highaffinity binding of aminoglycosides to prokaryotic rRNA, which has an adenine at position 1408 of the 16S rRNA (numbered according to the Escherichia coli sequence). In contrast, eucaryotic ribosomes have a guanine at the corresponding position, causing a low affinity towards aminoglycosides (Figure 2).11 The effects of aminoglycosides on protein translation in eucaryotic cells have been demonstrated at concentrations 10 to 15 times higher than the typical therapeutic antibacterial concentrations. Partial susceptibility of eucaryotic ribosomes toward aminoglycoside interaction has been traditionally viewed as the underlying mechanism of drug toxicity. However, this side effect may provide the opportunity for the treatment of human genetic diseases associated with translational defects.

AMINOGLYCOSIDES: PHARMACOGENETIC AGENTS

In 1985, Burke and Mogg12 were the first to demonstrate that the aminoglycoside antibiotics paromomycin and G-418 could partially restore the synthesis of a full-size protein from a mutant gene with a premature UAG mutation in cultured mammalian cells. Later, G-418 and gentamicin were shown to restore the expression of the cystic fibrosis transmembrane conductance regulator protein in a cell line carrying a nonsense mutation in cystic fibrosis transmembrane conductance regulator.13,14 In 1999, the ability of aminoglycosides to promote protein translation, despite the presence of a PTC mutation, was first demonstrated in vivo in a model of Duchenne muscular dystrophy.15 In this way, the concept of aminoglycoside-induced read-through emerged as a therapeutic strategy in human genetic disorders. In the current era, aminoglycosides have been shown to suppress premature translation termination at nonsense codons when bound to the rRNA decoding site interacting with codon–anticodon pairing in a series of clinically relevant conditions (Table 1). The efficiency of read-through varies from 1 percent to 25 percent in human cell lines, largely depending on the context of the stop mutation, with UGA showing greater translational read-through than UAG, and UAA exhibiting the most resistance to suppression.16,17 Several studies demonstrate the critical influence of upstream and downstream sequences on overall efficiency of translational termination.18,19 In mammalian cells, the action of the antibiotic is governed by nucleotides surrounding the stop mutation. The nucleotide in the position immediately downstream from the stop codon has a particularly significant impact on the efficiency of aminoglycoside-induced read-through, as follows: C4U4AXG.17 AMINOGLYCOSIDES: TRANSLATION OF PHARMACOGENETIC AGENTS IN MEDICINE

Figure 1 Structure of aminoglycoside bound to rRNA. Paromomycin (red) binds to helix 44 in 16S rRNA. Adenine in position 1408 is critical for high-affinity interaction with bacterial ribosome, with other residues critical for codon–anticodon cognition also indicated. Atomic coordinates are from 1IBK. 100

Production of protein in the presence of a nonsense PTC mutation resulting from aminoglycoside therapy, even if only in low amount, may be functionally significant. This is especially the case in recessive disorders, where protein expression is essentially absent. In such cases, even 1 percent of normal protein function may restore a near-normal or clinically less severe phenotype.2,4,6,7 Accordingly, it is primarily in recessive disorders that aminoglycosides have provided the greatest promise in both cell culture experiments and clinical trials. Examples include interventions with defective genes in cystic fibrosis, Duchenne muscular dystrophy, cystinosis, mucopolysaccharidosis type I (Hurler syndrome), X-linked nephrogenic diabetes insipidus, ataxia –telangiectasia, hemophilia, factor VII deficiency, and infantile neuronal ceroid lipofuscinosis (Table 1).20–29 Recent studies have indicated that read-through induction may be a promising therapy in autosomal-dominant disorders as well (Table 1).30–32 This is somewhat surprising since haploinsufficiency is the common pathogenic mechanism in nonsense mutation-related dominant disorders, i.e., B50 percent decrease in the amount of protein produced VOLUME 81 NUMBER 1 | JANUARY 2007 | www.nature.com/cpt

DISCOVERY

Figure 2 Aminoglycoside interaction with ribosomal protein translation. (a–c) Interaction of paromomycin with 16S rRNA of the 30S ribosome subunit. a In the absence of antibiotic, the conserved 16S rRNA (yellow strands) nucleotides A1492 and A1493 are stacked in the interior of helix 44. b Binding of mRNA codon and cognate tRNA causes conformational change of A1492, A1493, and G530 to accommodate energetically favorable interactions of ribosome components, tRNA and mRNA. c Paromomycin binding to the interior of helix44 induces a local conformational change in nucleotides A1492 and A1493, flipping them out. These changes facilitate binding of near-cognate tRNA. Black lines represent H-bonds. Crystal atomic coordinates are from 1J5E, IBK, and 1IBM. (d–f) Aminoglycoside induced read-through of the premature E375X stop codon in Kv1.5. d Normally, tRNA carrying glutamic acid (E) matches the mRNA codon to process Kv1.5 polypeptide elongation. Matching of the mRNA codon to the proper tRNA anticodon results in conformational alignment of A1492 and A1493 in the ribosomal decoding center (red dashes) and polypeptide chain elongation. e E375X (UAA codon in mRNA) mutation prevents codon–anticodon pairing and excludes the possibility of the A1492 and A1493 alignment in the ribosomal decoding center (red dashes) terminating protein translation. f Aminoglycoside binding to 16S rRNA induces conformational alignment in the ribosomal decoding center despite codon/anticodon mismatch. In the presence of aminoglycosides, the UAA codon may be paired with CUU or GUU tRNA anticodon promoting polypeptide chain elongation with glutamate or glutamine.

due to the heterozygous loss of one allele causes clinical symptoms. Just as a mutated protein can have a dominantnegative effect on the protein produced by the normal allele, even a small increase in full-length functional protein resulting from aminoglycoside therapy may improve outcome.30–32 A case in point is Hailey–Hailey disease, caused by a nonsense mutation in the Ca2 þ -ATPase2C1 transporter gene (ATP2C1),32 and familial atrial fibrillation related to a PTC mutation in KCNA5 encoding the voltage-sensitive potassium channel Kv1.5.31 In both cases, aminoglycosides have been suggested as corrective therapy.30,31 A limitation to aminoglycoside use as pharmacogenetic agents is necessary to reach concentrations sufficient to interfere with protein synthesis at human ribosomes, increasing risk of significant toxicity. Therefore, clinical success to date has been observed with local aminoglycoside delivery, particularly treatment of cystic fibrosis, the most studied disease in the context of pharmacogenetic-based therapy. Systemic delivery of currently available aminoglycosides at clinically approved doses has failed, as in the case of McArdle disease and Duchenne muscular dystrophy.33,34 Thus, developing effective drug delivery strategies that would minimize side effects and optimize safely achievable therapeutic levels is warranted. For example, percutaneous drugCLINICAL PHARMACOLOGY & THERAPEUTICS | VOLUME 81 NUMBER 1 | JANUARY 2007

eluted patch atrial implantation could be considered for patients with PTC mutations causing atrial arrhythmias. Furthermore, after treatment with aminoglycosides, enhanced production of functional protein from defective genes can be boosted through simultaneous application of protein-specific promoters.35 Also, recent discoveries in the structure of ribosomes and their interactions with aminoglycosides provide a platform on which to engineer new generations of pharmacogenetic agents with tissue predilection and molecular specificity, and limit or prevent undesired interactions with normal protein synthesis and function.9–11 CONCLUSION

Currently, aminoglycosides are the only clinically available drug family known to consistently affect gene translation in eucaryotes. This property has provided the foundation for preclinical and clinical testing of aminoglycosides to rescue protein production arrested by nonsense mutations. Aminoglycosides have shown initial promise in diverse genetic disorders due to PTC mutations, particularly when applied locally to secure sufficient therapeutic levels while avoiding systemic toxicity. The challenge in the emerging field of pharmacogenetic therapy will be to define the optimal strategy that would prevent interference with the normal 101

DISCOVERY Table 1 Genetic diseases and aminoglycoside-based pharmocogenetic therapy Disease

Affected gene

Ataxia–telangiectasia

ATM

Cystic fibrosis

CFTR

Model

Treatment outcome

Reference

In vitro cDNA-coupled transcription

Full-length functional protein

22

Cell culture


2,4,6,7,13,14,20,26

Transgenic mice

Amelioration of phenotype

Patients

Amelioration of clinical symptoms

Cell culture

Cystinosis

CTNS

Cell culture


Duchenne muscular dystrophy

DMD

Cell culture

Variable

23 2,7,15,17,24,34

Transgenic mice Patients Factor VII deficiency

F7

Cell culture


28

Familial atrial fibrillation

KCNA5

Cell culture


31

Hailey–Hailey disease

ATP2C1

Cell culture

Full-length protein synthesis

30

Hemophilia

F8 and F9

Patients


27

Infantile neuronal ceroid lipofuscinosis

TPP1

Cell culture

full-length functional protein

25

McArdle disease

PYGM

Patients

No significant benefit

Mucopolysaccharidosis Type 1 (Hurler syndrome)

IDUA

33


16,21

Cell culture


29

Transgenic mice

Amelioration of phenotype

In vitro cDNA-coupled transcription Cell culture

X-linked nephrogenic diabetes insipidus

AVPR2

protein synthesis process. In this regard, development of new drugs based on deciphering the intimate structural and functional determinants of aminoglycoside interaction with ribosomes, as well as the establishment of new modalities for targeted drug delivery are priorities. Indeed, a better understanding of the benefits afforded by combinatorial use of aminoglycosides with gene-of-interest-specific promoters, enhanced local application, as well as determining the most favorable schedule for long-term therapy will be critical for the future of this promising strategy for gene code repair in otherwise untreatable genetic diseases.

4. 5.

6.

7.

8.

9.

ACKNOWLEDGMENTS LVZ is the recipient of the Kogod Program on Aging Career Development Award. This work was supported by grants from the National Institutes of Health, Marriott Heart Disease Research Program, Marriott Foundation, Ted Nash Long Life Foundation, and Ralph Wilson Medical Research Foundation. CONFLICT OF INTEREST The authors declared no conflict of interest.

10. 11. 12.

13.

14. & 2007 American Society for Clinical Pharmacology and Therapeutics

1. 2. 3.

102

Nirenberg, M. The genetic code: Nobel lecture, http://nobelprize.org/ nobel_prizes/medicine/laureates/1968/press.html (1968). Kellermayer, R. Translational readthrough induction of pathogenic nonsense mutations. Eur. J. Med. Genet. (2006). Kusik, V. Mendelian Inheritance in Man: A Catalog of Human Genes and Genetic Disorders, (John Hopkins University Press: Baltimore, 1998).

15.

16.

Byers, P.H. Killing the messenger: new insights into nonsense-mediated mRNA decay. J. Clin. Invest. 109, 3–6 (2002). Roberts, R.G., Gardner, R.J. & Bobrow, M. Searching for the 1 in 2,400,000: a review of dystrophin gene point mutations. Hum. Mutat. 4, 1–11 (1994). Lukacs, G.L. & Durie, P.R. Pharmacologic approaches to correcting the basic defect in cystic fibrosis. N. Engl. J. Med. 349, 1401–1404 (2003). Holbrook, J.A., Neu-Yilik, G., Hentze, M.W. & Kulozik, A.E. Nonsense-mediated decay approaches the clinic. Nat. Genet. 36, 801–808 (2004). Luzzatto, L., Apirion, D. & Schlessinger, D. Mechanism of action of streptomycin in E. coli: interruption of the ribosome cycle at the initiation of protein synthesis. Proc. Natl. Acad. Sci. USA 60, 873–880 (1968). Yoshizawa, S., Fourmy, D. & Puglisi, J.D. Structural origins of gentamicin antibiotic action. EMBO J. 17, 6437–6448 (1998). Ogle, J.M. et al. Recognition of cognate transfer RNA by the 30S ribosomal subunit. Science 292, 897–902 (2001). Recht, M.I., Douthwaite, S. & Puglisi, J.D. Basis for prokaryotic specificity of action of aminoglycoside antibiotics. EMBO J. 18, 3133–3138 (1999). Burke, J.F. & Mogg, A.E. Suppression of a nonsense mutation in mammalian cells in vivo by the aminoglycoside antibiotics G-418 and paromomycin. Nucleic Acids Res. 13, 6265–6272 (1985). Howard, M., Frizzell, R.A. & Bedwell, D.M. Aminoglycoside antibiotics restore CFTR function by overcoming premature stop mutations. Nat. Med. 2, 467–469 (1996). Bedwell, D.M. et al. Suppression of a CFTR premature stop mutation in a bronchial epithelial cell line. Nat. Med. 3, 1280–1284 (1997). Barton-Davis, E.R., Cordier, L., Shoturma, D.I., Leland, S.E. & Sweeney, H.L. Aminoglycoside antibiotics restore dystrophin function to skeletal muscles of mdx mice. J. Clin. Invest. 104, 375–381 (1999). Keeling, K.M. & Bedwell, D.M. Clinically relevant aminoglycosides can suppress disease-associated premature stop mutations in the IDUA and P53 cDNAs in a mammalian translation system. J. Mol. Med. 80, 367–376 (2002). VOLUME 81 NUMBER 1 | JANUARY 2007 | www.nature.com/cpt

DISCOVERY 17. Howard, M.T. et al. Sequence specificity of aminoglycoside-induced stop condon readthrough: potential implications for treatment of Duchenne muscular dystrophy. Ann. Neurol. 48, 164–169 (2000). 18. Bonetti, B., Fu, L., Moon, J. & Bedwell, D.M. The efficiency of translation termination is determined by a synergistic interplay between upstream and downstream sequences in Saccharomyces cerevisiae. J. Mol. Biol. 251, 334–345 (1995). 19. Namy, O., Hatin, I. & Rousset, J.P. Impact of the six nucleotides downstream of the stop codon on translation termination. EMBO Rep. 2, 787–793 (2001). 20. Clancy, J.P. et al. Evidence that systemic gentamicin suppresses premature stop mutations in patients with cystic fibrosis. Am. J. Respir. Crit. Care Med. 163, 1683–1692 (2001). 21. Hein, L.K. et al. Alpha-L-iduronidase premature stop codons and potential read-through in mucopolysaccharidosis type I patients. J. Mol. Biol. 338, 453–462 (2004). 22. Lai, C.H. et al. Correction of ATM gene function by aminoglycosideinduced read-through of premature termination codons. Proc. Natl. Acad. Sci. USA 101, 15676–15681 (2004). 23. Helip-Wooley, A., Park, M.A., Lemons, R.M. & Thoene, J.G. Expression of CTNS alleles: subcellular localization and aminoglycoside correction in vitro. Mol. Genet. Metab. 75, 128–133 (2002). 24. Politano, L. et al. Gentamicin administration in Duchenne patients with premature stop codon. Preliminary results. Acta Myol. 22, 15–21 (2003). 25. Sleat, D.E., Sohar, I., Gin, R.M. & Lobel, P. Aminoglycoside-mediated suppression of nonsense mutations in late infantile neuronal ceroid lipofuscinosis. Eur. J. Paediatr. Neurol. 5, 57–62 (2001).

CLINICAL PHARMACOLOGY & THERAPEUTICS | VOLUME 81 NUMBER 1 | JANUARY 2007

26. Wilschanski, M. et al. Gentamicin-induced correction of CFTR function in patients with cystic fibrosis and CFTR stop mutations. N. Engl. J. Med. 349, 1433–1441 (2003). 27. James, P.D. et al. Aminoglycoside suppression of nonsense mutations in severe hemophilia. Blood 106, 3043–3048 (2005). 28. Pinotti, M. et al. Intracellular readthrough of nonsense mutations by aminoglycosides in coagulation factor VII. J. Thromb. Haemost. 4, 1308–1314 (2006). 29. Sangkuhl, K. et al. Aminoglycoside-mediated rescue of a disease-causing nonsense mutation in the V2 vasopressin receptor gene in vitro and in vivo. Hum. Mol. Genet. 13, 893–903 (2004). 30. Kellermayer, R., Szigeti, R., Keeling, K.M., Bedekovics, T. & Bedwell, D.M. Aminoglycosides as potential pharmacogenetic agents in the treatment of Hailey–Hailey disease. J. Invest. Dermatol. 126, 229–231 (2006). 31. Olson, T.M. et al. Kv1.5 channelopathy due to KCNA5 loss-of-function mutation causes human atrial fibrillation. Hum. Mol. Genet. 15, 2185–2191 (2006). 32. Porgpermdee, S. et al. Expression of SPCA1 (Hailey–Hailey disease gene product) in acantholytic dermatoses. J. Dermatol. Sci. 40, 137–140 (2005). 33. Schroers, A. et al. Gentamicin treatment in McArdle disease: failure to correct myophosphorylase deficiency. Neurology 66, 285–286 (2006). 34. Wagner, K.R. et al. Gentamicin treatment of Duchenne and Becker muscular dystrophy due to nonsense mutations. Ann. Neurol. 49, 706–711 (2001). 35. Xi, B., Guan, F. & Lawrence, D.S. Enhanced production of functional proteins from defective genes. J. Am. Chem. Soc. 126, 5660–5661 (2004).

103