1. I DENTIFICATION OF CHEMICAL AND PHARMACOLOGICAL CHAPERONES TO TREAT ZSD PATIENTS WITH THE COMMON ALLELE, PEX1- GLY 843 ASP Gillian MacLean, Braverman Laboratory McGill University, Department of Human Genetics GFPD Family & Scientific Conference Lincoln, Nebraska July 28, 2013

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

3. P EROXISOME B IOGENESIS D ISORDERS 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. P EX PROTEINS ARE INVOLVED IN PEROXISOME MATRIX ENZYME IMPORT

5. P ROTEIN 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. M OST 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-G LY 843A SP (G843D) Missense allele Misfolded protein Increased degradation Reduced function However: Milder affect on patients Progressive Glycine (G) Aspartate (D) Arakawa et al. 2006 Non-native Unfolded protein Intermediate Native protein Mutation

8. C ELL 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

9. Courtesy of Joe Hacia

10. F UNCTIONAL RECOVERY OF PEROXISOMES OBSERVED IN TREATED PEX1-G843D FIBROBLASTS GFP-PTS1 reporter localizes to the peroxisomes when: Grown at lower temperatures Grown with non- specific chemical chaperones (Zhang et al., 2010) 30 O CUntreated 200 mM TMAO100 mM betaine

11. F UNCTIONAL 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. A SSAY EFFECTIVELY USED FOR THE IDENTIFICATION OF POTENTIAL DRUGS Screened 2000 small molecule compounds Identified hit compounds  flavonoids No treatment (- ) 150 mM Betaine(+)10 uM Diosmetin

13. T ESTING OF ADDITIONAL FLAVONOIDS Tested >50 flavonoids Compared import recovery by dose response

14. D ISCOVERY 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. P OTENTIAL FOR COMBINATION THERAPIES Chemical chaperones: Interact with proteins non-selectively  Betaine Pharmacological chaperones: Interact with proteins selectively  Flavonoids Proteasome inhibitors: Inhibit degradation of misfolded proteins  Bortezomib improve PEX1-G843D folding PEX1-G843D levels

16. C OMBINATION THERAPIES RESULT IN AN ADDITIVE EFFECT Low doses betaine + flavonoid = more effective than high dose flavonoid

17. C ONFIRMATION OF CELL - BASED REPORTER ASSAY Evaluate recovery of endogenous matrix enzyme import Evaluate biochemical parameters Plasmalogen levels DHA levels

18. S UMMARY AND F UTURE D IRECTIONS

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