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
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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
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
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Proteins

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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
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60 % of all ZSD alleles
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20% = PEX1-c.2097_2098insT (p.Ile700fs) (null)
20-30% = PEX1-c.2528G>A (p.Gly843Asp) (missense)
PEX1-GLY843ASP (G843D)

Missense allele
Aspartate (D)
Glycine (G)

Misfolded protein
Increased degradation
 Reduced function

Non-native

Mutation
Unfolded
protein
However:
Milder affect on patients
 Progressive

Intermediate
Arakawa et al. 2006
Native
protein
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
Courtesy of Joe Hacia
FUNCTIONAL RECOVERY OF PEROXISOMES OBSERVED
IN TREATED PEX1-G843D FIBROBLASTS
Untreated
30 OC

200 mM TMAO
GFP-PTS1 reporter
localizes to the
peroxisomes when:

Grown at lower
temperatures

Grown with nonspecific chemical
chaperones
100 mM betaine
(Zhang et al., 2010)
FUNCTIONAL RECOVERY SUGGESTS
IMPROVED FOLDING


Decrease temperature
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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 (- )
10 uM Diosmetin
150 mM Betaine(+)
TESTING OF ADDITIONAL FLAVONOIDS
Tested >50 flavonoids
 Compared import recovery
by dose response
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2,5, and blinded comb
% Importing Cells
100
Diosmetin
Acacetin
Ac. diacetate
Apigenin
Kaempferol
Chrysin
Galangin
Tamarixitin
80
60
40
20
0
0
10
20
Concentration (M)
30
DISCOVERY OF POTENTIAL
PHARMACOLOGICAL CHAPERONES
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Pharmacological chaperones:
Interact with proteins selectively -> stabilize or improve folding
 May be, or mimic binding partners
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Enzyme substrate
Protein ligand
Co-factors
PEX1 is a
AAA ATPase
Flavonoids bind
ATP bining sites
POTENTIAL FOR COMBINATION THERAPIES
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Chemical chaperones:
Interact with proteins non-selectively
 Betaine
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Pharmacological chaperones:
improve
PEX1-G843D
folding
Interact with proteins selectively
 Flavonoids


Proteasome inhibitors:
Inhibit degradation of misfolded proteins
 Bortezomib

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
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SUMMARY AND FUTURE DIRECTIONS

Effective cell based assay
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PEX1-G843D patient cell line
GFP-PTS1 reporter
Demonstrates recovery
Chemical and pharmacological chaperones identified
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Shown to work in combination
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Better understand current potential compounds
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Develop more sensitive, more general assays
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Continue to look for even better compounds
Treat a broader group of patients
ACKNOWLEDGEMENTS
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Nancy Braverman laboratory
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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
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A special thanks to
families and patients for
their kind contributions
FUNDING ORGANIZATIONS

Woodbury Peroxisome Disease Family Funding