i dentification of chemical and pharmacological chaperones to treat zsd patients with the common...
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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