PTC readthrough opportuni1es for RDEB therapy: novel candidate drugs

Transcription

1 PTC readthrough opportuni1es for RDEB therapy: novel candidate drugs Michel Roberge Biochemistry & Molecular Biology University of Bri1sh Columbia Canada 1

2 11% 10-20% for EB 30% Die before 5 th birthday 95% Have no treatment Nonsense muta+ons Single base subs1tu1ons that introduce a premature termina1on codon (PTC) Therapies directed at nonsense muta1ons could benefit many pa1ents across many gene1c disorders

3 Induc1on of full- length protein by premature termina1on codon (PTC) readthrough mrna protein CGA Arg UGA stop AAAA WT full- length protein func1onal mrna protein UGA ptc UGA stop AAAA PTC no full length protein low levels of mrna (NMD) and truncated protein mrna protein UGA ptc near- cognate AA UGA stop AAAA PTC readthrough full- length protein largely ac1ve 3

4 Part 1 A new look at gentamicin Clinical trials for cys1c fibrosis and Duchenne muscular dystrophy (ca 2000) Improvements were observed but they were small and variable Different results may be avributable to different brands of gentamicin used in the mouse and human studies cited Karpa% and Lochmuller Ann. Neurol. 49:693 (2001) The variable response found among different studies in CF pa1ents, DMD mice models and DMD pa1ents might be avributed to different brands, which might contain different rela1ve concentra1on of each gentamicin component Linde and Kerem Trends Genet. 24:552 (2008) The source of the an1bio1c could have an influence on efficacy In commercial produc1on, a par1cular mixture of these three isoforms (C1, C1a, C2) could be more or less effec1ve in the laboratory or in the clinic. For clinical trials, it is important to use gentamicin from the same source Malik et al. Ther. Adv. Neurol. Dis. 3:379 (2010) Given that gentamicin has variable effects and exhibits some toxicity, less toxic effec1ve deriva1ves of this drug need to be developed for an effec1ve DMD treatment Pichavant et al. Mol. Ther. 19:830 (2011) 4

5 Pharmaceu1cal gentamicin is not a pure compound USP 25-50% 10-35% 25-55% 5

6 Model system: HDQ- P1 breast cancer cell line with homozygous TP53 nonsense muta1on Automated p53 western assay for readthrough Pseudoblot representa1on Readthrough R213X 6

7 Gentamicin batches show variable readthrough ac1vity Major components (>97%) Minor components (<3%) R 2 R 1 H 2 N H H H NH 3 C NH2 H CH 3 H NH2 H H H H NH2 H CH 3 H NH 3 C H H 2 N H NH2 H CH 3 H 3 C NH H H 3 C H H H H H NH2 H H CH 3 H NH 3 C H H NH2 H H CH 3 H NH 3 C H 7

8 Major gentamicin components are inac1ve Readthrough Readthrough Gentamicin B1 shows potent PTC readthrough ac1vity H 3 C H H H H NH2 H CH 3 H 3 C NH H 8

9 Ac1vity in cells from rare gene1c disease pa1ents Duchenne muscular dystrophy Neuronal ceroid lipofuscinosis CLN2 Schimke immuno- osseous dysplasia Recessive dystrophic epidermolysis bullosa 9

10 Part 2 Enhancers of PTC readthrough by aminoglycosides H N Cl HN Cl CDX > 200 analogs > CDX5-1 Small molecules that do not themselves induce readthrough but poten1ate readthrough by aminoglycosides 10

11 Time course CDX5-1 + G418 11

12 Time course CDX5-1 + G418 Compounds also poten1ate readthrough by gentamicin B1 12

13 Ac1vity in cells from rare gene1c disease pa1ents - Neuronal ceroid lipofuscinosis (lysosomal storage disease) - Muta1ons in the CLN2 gene encoding tripep1dylpep1dase 1 (TPP1) - Primary fibroblasts from a pa1ent with nonsense muta1ons (R127X/R208X) Ala- Ala- Phe- 7- amido- 4- methylcoumarin 13

14 Summary - Gentamicin B1: a minor gentamicin that potently induces PTC readthrough - Four dis1nct structural classes of small molecules that poten1ate PTC readthrough by aminoglycosides Combina1on of B1 and a poten1ator may broadly suppress nonsense muta1ons in a variety of gene1c diseases including EB 14

15 Summary - Gentamicin B1: a minor gentamicin that potently induces PTC readthrough - Four dis1nct structural classes of small molecules that poten1ate PTC readthrough by aminoglycosides Combina1on of B1 and a poten1ator may broadly suppress nonsense muta1ons in a variety of gene1c diseases including EB New collabora1ons with Cris1na Has, Fernando Larcher and Andrew South First EB grant, star1ng Sept

16 Hilary Anderson Aruna Balgi Alireza Baradaran- Heravi Kunho Choi Mike Ferguson Carla Zimmerman CDRD Marcel Bally Nancy Dos Santos Dana Masin Juergen Niesser Natalie Strynadka Andrew South James Jaquith Tom Pfeifer Fahimeh Shidmoossavee Yoko Shimizu Jason Tan 16