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

OUTLINE

BackgroundPeroxisome matrix enzyme importProteins 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

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

PEX PROTEINS ARE INVOLVED IN PEROXISOME MATRIX ENZYME IMPORT

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

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)

PEX1-GLY843ASP (G843D)

Missense allele

Misfolded protein Increased degradation Reduced function

However: Milder affect on patients Progressive

O

OH

NH2NH2O

HO

O

OH

Glycine (G) Aspartate (D)

Arakawa et al. 2006

Non-native

Unfolded protein

Intermediate

Native protein

Mutation

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

Courtesy of Joe Hacia

FUNCTIONAL 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 OCUntreated

200 mM TMAO 100 mM betaine

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

ASSAY 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

TESTING OF ADDITIONAL FLAVONOIDS

Tested >50 flavonoids Compared import recovery by dose response

2,5, and blinded comb

0 10 20 300

20

40

60

80

100Diosmetin

Acacetin

Ac. diacetate

Apigenin

Kaempferol

Chrysin

Galangin

Tamarixitin

Concentration (M)

% Im

po

rtin

g C

ells

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

POTENTIAL 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

COMBINATION THERAPIES RESULT IN AN ADDITIVE EFFECT

Low doses betaine + flavonoid = more effective than high dose flavonoid

CONFIRMATION OF CELL-BASED REPORTER ASSAY

Evaluate recovery of endogenous matrix enzyme import

Evaluate biochemical parameters Plasmalogen levels DHA levels IN PROGRESS

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!

ACKNOWLEDGEMENTS

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

FUNDING ORGANIZATIONS Woodbury Peroxisome Disease Family Funding

A special thanks to families and patients for their kind contributions