Gillian MacLean, Braverman Laboratory McGill University, Department of Human Genetics

1. Identification of chemical and pharmacological chaperones to treat ZSD patients with the common allele, PEX1-gly843asp Gillian MacLean, Braverman Laboratory McGill University, Department of Human Genetics GFPD Family & Scientific Conference Lincoln, Nebraska July 28, 2013

2. Outline • Background • Peroxisome matrix enzyme import • Proteins and impact of genetic changes • PEX1 null and missense mutations • Development of cell based assay • Identification of drugs • chemical, pharmacological, combination therapies • Future directions

3. Peroxisome Biogenesis Disorders • Zellweger spectrum disorder (ZSD) (~1/60,000) • Zellweger syndrome • Neonatal adrenoleukodystrophy • Infantile refsum disease • Cannot assemble normal peroxisomes • Multiple enzyme deficiencies • Mutations in PEX genes lead to defects in PEX proteins • Broad spectrum  Can relate to which protein is affected and what the mutation is

4. Pex proteins are involved in peroxisome matrix enzyme import

5. Protein synthesis depends on DNA sequence • Proteins • Polypeptides comprised of linked of amino acids • Linear sequence gives rise to folded protein • Sequence encoded by DNA • Null allele = • no protein produced • Missense allele = • different amino acid incorporated Nature Education, 2010

6. Most ZSD mutations are associated with the PEX1 gene • Encodes the PEX1 protein • AAA ATPase (ATPase associated • with diverse cellular activities) • Uses energy from ATP to recycle • PEX5 for additional rounds of import • 60 % of all ZSD alleles • 20% = PEX1-c.2097_2098insT (p.Ile700fs) (null) • 20-30% = PEX1-c.2528G>A (p.Gly843Asp) (missense)

7. PEX1-Gly843Asp (G843D) • Missense allele • Misfolded protein • Increased degradation • Reduced function • However: • Milder affect on patients • Progressive Aspartate (D) Glycine (G) Non-native Mutation Unfolded protein Native protein Intermediate Arakawa et al. 2006

8. Cell based assay developed to detect recovery of reporter protein importation • Patient fibroblasts grown in cell culture • expresses “Green Fluorescent Protein” (GFP)-PTS1 reporter • PEX1-G843D/null G843D

9. Courtesy of Joe Hacia

10. Functional recovery of peroxisomes observed in treated PEX1-G843D fibroblasts Untreated 30 OC • GFP-PTS1 reporter localizes to the peroxisomes when: • Grown at lower temperatures • Grown with non-specific chemical chaperones 200 mM TMAO 100 mM betaine (Zhang et al., 2010)

11. Functional recovery suggests improved folding • Decrease temperature • Cells are in lower energy state • Reduced degradation of misfolded proteins • Proteins have more time to find correct conformation • Not applicable for patients • Chemical chaperones • Create environment for better protein folding • Non- specifically enhances protein folding • Requires high concentrations

12. Assay effectively used for the identification of potential drugs • Screened 2000 small molecule compounds • Identified hit compounds flavonoids No treatment (- ) 10 uM Diosmetin 150 mM Betaine(+)

13. Testing of additional flavonoids Tested >50 flavonoids Compared import recovery by dose response

14. Discovery of potential pharmacological chaperones • Pharmacological chaperones: • Interact with proteins selectively -> stabilize or improve folding • May be, or mimic binding partners • Enzyme substrate • Protein ligand • Co-factors • PEX1 is a AAA ATPase • Flavonoids bind ATP bining sites

15. Potential for combination therapies improve PEX1-G843D folding • Chemical chaperones: • Interact with proteins non-selectively • Betaine • Pharmacological chaperones: • Interact with proteins selectively • Flavonoids • Proteasome inhibitors: • Inhibit degradation of misfolded proteins • Bortezomib PEX1-G843D levels

16. Combination therapies result in an additive effect • Low doses betaine + flavonoid = more effective than high dose flavonoid

17. Confirmation of cell-based reporter assay • Evaluate recovery of endogenous matrix enzyme import • Evaluate biochemical parameters • Plasmalogen levels • DHA levels IN PROGRESS

18. Summary and Future Directions • Effective cell based assay • PEX1-G843D patient cell line • GFP-PTS1 reporter • Demonstrates recovery • Chemical and pharmacological chaperones identified • Shown to work in combination • Great starting point! • Better understand current potential compounds • Develop more sensitive, more general assays • Continue to look for even better compounds • Treat a broader group of patients • Make the best and safest drugs available ASAP!

19. Acknowledgements • McGill community • Eric Shoubridge and Olga Zurita • Armando Jardim • Murielle Akpa • A special thanks to families and patients for their kind contributions • Nancy Braverman laboratory • Catherine Argyriou • Sara Birjandian and Tara Saberian • Sarn Jiralerspong • Erminia Di Pietro • Claudia Matos-Miranda • Wei Cui • Steve Steinberg and ShandiHiebler • Joe Hacia • Gabrielle Dodt Funding organizations • Woodbury Peroxisome Disease Family Funding