The novel aminoglycoside, ELX-02, permits CTNSW138X translational read-through and restores lysosomal cystine efflux in cystinosis

English version

Autoři: Emma J. Brasell ^aff001; Lee Lee Chu ^aff002; Murielle M. Akpa ^aff002; Idit Eshkar-Oren ^aff003; Iris Alroy ^aff003; Rachel Corsini ^aff002; Brian M. Gilfix ^aff002; Yojiro Yamanaka ^aff001; Pedro Huertas ^aff003; Paul Goodyer ^aff001
Působiště autorů: McGill University, Department of Human Genetics, Montreal, Canada ^aff001; Research Institute of the McGill University Health Centre, Montreal, Canada ^aff002; McGill University, Department of Experimental Medicine, Montreal, Canada ^aff003; Montreal Children’s Hospital, Department of Nephrology, Montreal, Canada ^aff004; Eloxx Pharmaceuticals, Inc., Waltham, United States of America ^aff005
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pone.0223954

Souhrn

Background

Cystinosis is a rare disorder caused by recessive mutations of the CTNS gene. Current therapy decreases cystine accumulation, thus slowing organ deterioration without reversing renal Fanconi syndrome or preventing eventual need for a kidney transplant.15-20% of cystinosis patients harbour at least one nonsense mutation in CTNS, leading to premature end of translation of the transcript. Aminoglycosides have been shown to permit translational read-through but have high toxicity level, especially in the kidney and inner ear. ELX-02, a modified aminoglycoside, retains it read-through ability without the toxicity.

Methods and findings

We ascertained the toxicity of ELX-02 in cells and in mice as well as the effect of ELX-02 on translational read-through of nonsense mutations in cystinotic mice and human cells. ELX-02 was not toxic in vitro or in vivo, and permitted read-through of nonsense mutations in cystinotic mice and human cells.

Conclusions

ELX-02 has translational read-through activity and produces a functional CTNS protein, as evidenced by reduced cystine accumulation. This reduction is comparable to cysteamine treatment. ELX-02 accumulates in the kidney but neither cytotoxicity nor nephrotoxicity was observed.

Klíčová slova:

Blood – Blood plasma – Mouse models – Fibroblasts – Kidneys – Toxicity – Nonsense mutation – Lysosomes

Zdroje

1. Kalatzis V, Cherqui S, Antignac C, Gasnier B. Cystinosin, the protein defective in cystinosis, is a H(+)-driven lysosomal cystine transporter. EMBO J. 2001;20(21):5940–9. doi: 10.1093/emboj/20.21.5940 11689434.

2. Nesterova G, Williams C, Bernardini I, Gahl WA. Cystinosis: renal glomerular and renal tubular function in relation to compliance with cystine-depleting therapy. Pediatric nephrology. 2014. Epub 2014/12/21. doi: 10.1007/s00467-014-3018-x 25526929.

3. Midgley JP, El-Kares R, Mathieu F, Goodyer P. Natural history of adolescent-onset cystinosis. Pediatric nephrology. 2011;26(8):1335–7. Epub 2011/05/10. doi: 10.1007/s00467-011-1904-z 21553323.

4. Thoene JG, Oshima RG, Crawhall JC, Olson DL, Schneider JA. Cystinosis. Intracellular cystine depletion by aminothiols in vitro and in vivo. The Journal of clinical investigation. 1976;58(1):180–9. doi: 10.1172/JCI108448 932205.

5. Gahl WA, Reed GF, Thoene JG, Schulman JD, Rizzo WB, Jonas AJ, et al. Cysteamine therapy for children with nephropathic cystinosis. The New England journal of medicine. 1987;316(16):971–7. doi: 10.1056/NEJM198704163161602 3550461.

6. Brodin-Sartorius A, Tete MJ, Niaudet P, Antignac C, Guest G, Ottolenghi C, et al. Cysteamine therapy delays the progression of nephropathic cystinosis in late adolescents and adults. Kidney international. 2012;81(2):179–89. Epub 2011/09/09. doi: 10.1038/ki.2011.277 21900880.

7. Ivanova EA, De Leo MG, Van Den Heuvel L, Pastore A, Dijkman H, De Matteis MA, et al. Endo-lysosomal dysfunction in human proximal tubular epithelial cells deficient for lysosomal cystine transporter cystinosin. PloS one. 2015;10(3):e0120998. Epub 2015/03/27. doi: 10.1371/journal.pone.0120998 25811383.

8. Taranta A, Petrini S, Palma A, Mannucci L, Wilmer MJ, De Luca V, et al. Identification and subcellular localization of a new cystinosin isoform. American journal of physiology Renal physiology. 2008;294(5):F1101–8. Epub 2008/03/14. doi: 10.1152/ajprenal.00413.2007 18337546.

9. Saudek V. Cystinosin, MPDU1, SWEETs and KDELR belong to a well-defined protein family with putative function of cargo receptors involved in vesicle trafficking. PloS one. 2012;7(2):e30876. Epub 2012/03/01. doi: 10.1371/journal.pone.0030876 22363504.

10. Forestier L, Jean G, Attard M, Cherqui S, Lewis C, van’t Hoff W, et al. Molecular characterization of CTNS deletions in nephropathic cystinosis: development of a PCR-based detection assay. American journal of human genetics. 1999;65(2):353–9. Epub 1999/07/27. doi: 10.1086/302509 10417278.

11. McGowan-Jordan J, Stoddard K, Podolsky L, Orrbine E, McLaine P, Town M, et al. Molecular analysis of cystinosis: probable Irish origin of the most common French Canadian mutation. European journal of human genetics: EJHG. 1999;7(6):671–8. doi: 10.1038/sj.ejhg.5200349 10482956.

12. Heier CR, DiDonato CJ. Translational readthrough by the aminoglycoside geneticin (G418) modulates SMN stability in vitro and improves motor function in SMA mice in vivo. Hum Mol Genet. 2009;18(7):1310–22. Epub 2009/01/20. doi: 10.1093/hmg/ddp030 19150990.

13. Yu H, Liu X, Huang J, Zhang Y, Hu R, Pu J. Comparison of read-through effects of aminoglycosides and PTC124 on rescuing nonsense mutations of HERG gene associated with long QT syndrome. International journal of molecular medicine. 2014;33(3):729–35. Epub 2013/12/25. doi: 10.3892/ijmm.2013.1601 24366185.

14. Shulman E, Belakhov V, Wei G, Kendall A, Meyron-Holtz EG, Ben-Shachar D, et al. Designer aminoglycosides that selectively inhibit cytoplasmic rather than mitochondrial ribosomes show decreased ototoxicity: a strategy for the treatment of genetic diseases. The Journal of biological chemistry. 2014;289(4):2318–30. Epub 2013/12/05. doi: 10.1074/jbc.M113.533588 24302717.

15. Kandasamy J, Atia-Glikin D, Shulman E, Shapira K, Shavit M, Belakhov V, et al. Increased selectivity toward cytoplasmic versus mitochondrial ribosome confers improved efficiency of synthetic aminoglycosides in fixing damaged genes: a strategy for treatment of genetic diseases caused by nonsense mutations. Journal of medicinal chemistry. 2012;55(23):10630–43. Epub 2012/11/15. doi: 10.1021/jm3012992 23148581 mc3542475.

16. Sabbavarapu NM, Shavit M, Degani Y, Smolkin B, Belakhov V, Baasov T. Design of Novel Aminoglycoside Derivatives with Enhanced Suppression of Diseases-Causing Nonsense Mutations. ACS Med Chem Lett. 2016;7(4):418–23. Epub 2016/04/21. doi: 10.1021/acsmedchemlett.6b00006 27096052.

17. Brasell EJ, Chu L, El Kares R, Seo JH, Loesch R, Iglesias DM, et al. The aminoglycoside geneticin permits translational readthrough of the CTNS W138X nonsense mutation in fibroblasts from patients with nephropathic cystinosis. Pediatric nephrology. 2018. Epub 2018/11/11. doi: 10.1007/s00467-018-4094-0 30413946.

18. Gilfix BM, Blank DW, Rosenblatt DS. Novel reductant for determination of total plasma homocysteine. Clinical chemistry. 1997;43(4):687–8. Epub 1997/04/01. 9105275.

19. Cherqui S, Sevin C, Hamard G, Kalatzis V, Sich M, Pequignot MO, et al. Intralysosomal cystine accumulation in mice lacking cystinosin, the protein defective in cystinosis. Mol Cell Biol. 2002;22(21):7622–32. doi: 10.1128/MCB.22.21.7622-7632.2002 12370309.

20. Emma F, Nesterova G, Langman C, Labbe A, Cherqui S, Goodyer P, et al. Nephropathic cystinosis: an international consensus document. Nephrology, dialysis, transplantation: official publication of the European Dialysis and Transplant Association—European Renal Association. 2014;29(suppl 4):iv87–iv94. Epub 2014/08/29. doi: 10.1093/ndt/gfu090 25165189 mc4158338.

21. Jezegou A, Llinares E, Anne C, Kieffer-Jaquinod S, O’Regan S, Aupetit J, et al. Heptahelical protein PQLC2 is a lysosomal cationic amino acid exporter underlying the action of cysteamine in cystinosis therapy. Proc Natl Acad Sci U S A. 2012;109(50):E3434–43. Epub 2012/11/22. doi: 10.1073/pnas.1211198109 23169667.

22. Zhang J, Wang J, Ng S, Lin Q, Shen HM. Development of a novel method for quantification of autophagic protein degradation by AHA labeling. Autophagy. 2014;10(5):901–12. Epub 2014/03/29. doi: 10.4161/auto.28267 24675368.

23. Varshavsky A. The N-end rule pathway of protein degradation. Genes to cells: devoted to molecular & cellular mechanisms. 1997;2(1):13–28. Epub 1997/01/01. doi: 10.1046/j.1365-2443.1997.1020301.x 9112437.

24. Manuvakhova M, Keeling K, Bedwell DM. Aminoglycoside antibiotics mediate context-dependent suppression of termination codons in a mammalian translation system. Rna. 2000;6(7):1044–55. Epub 2000/08/05. doi: 10.1017/s1355838200000716 10917599.

25. Schneider JA, Bradley K, Seegmiller JE. Increased cystine in leukocytes from individuals homozygous and heterozygous for cystinosis. Science. 1967;157(3794):1321–2. doi: 10.1126/science.157.3794.1321 6038997.

26. Gahl WA, Tietze F, Bashan N, Steinherz R, Schulman JD. Defective cystine exodus from isolated lysosome-rich fractions of cystinotic leucocytes. The Journal of biological chemistry. 1982;257(16):9570–5. 7107582.

27. Kalatzis V, Nevo N, Cherqui S, Gasnier B, Antignac C. Molecular pathogenesis of cystinosis: effect of CTNS mutations on the transport activity and subcellular localization of cystinosin. Hum Mol Genet. 2004;13(13):1361–71. doi: 10.1093/hmg/ddh152 15128704.

28. Lee HL, Dougherty JP. Pharmaceutical therapies to recode nonsense mutations in inherited diseases. Pharmacology & therapeutics. 2012;136(2):227–66. Epub 2012/07/24. doi: 10.1016/j.pharmthera.2012.07.007 22820013.

29. Sun J, Chen M, Xu J, Luo J. Relationships among stop codon usage bias, its context, isochores, and gene expression level in various eukaryotes. Journal of molecular evolution. 2005;61(4):437–44. Epub 2005/09/20. doi: 10.1007/s00239-004-0277-3 16170455.

30. Ivanova EA, van den Heuvel LP, Elmonem MA, De Smedt H, Missiaen L, Pastore A, et al. Altered mTOR signalling in nephropathic cystinosis. Journal of inherited metabolic disease. 2016;39(3):457–64. Epub 2016/02/26. doi: 10.1007/s10545-016-9919-z 26909499.

31. Shotelersuk V, Larson D, Anikster Y, McDowell G, Lemons R, Bernardini I, et al. CTNS mutations in an American-based population of cystinosis patients. American journal of human genetics. 1998;63(5):1352–62. Epub 1998/10/30. doi: 10.1086/302118 9792862.

32. Town M, Jean G, Cherqui S, Attard M, Forestier L, Whitmore SA, et al. A novel gene encoding an integral membrane protein is mutated in nephropathic cystinosis. Nature genetics. 1998;18(4):319–24. doi: 10.1038/ng0498-319 9537412.