GFPD FAMILY AND SCIENTIFIC CONFERENCE
DEFINING HOW DIOSMETIN WORKS
FOR PEX1-GLY483ASP AND
PARTNERING WITH DRUG COMPANIES
July 28th, 2013
Lincoln, Nebrasaka
By Catherine Argyriou, Braverman laboratory
McGill University Department of Human Genetics
SUMMARY
1.
The function of PEX1
a. The importance of PEX1
b. The PEX1-G843D allele
c. Effect of PEX1 misfolding
2.
Improving the function of PEX1-G843D
a. Pharmacologic chaperone- diosmetin
b. Proposed mechanism of action
c. Designing the optimal drug
d. Pharmaceutical company collaboration
3.
The path forward
a. Defining how diosmetin and other
pharmacologic chaperones work
b. Pharmacologic chaperones and other
mutations
THE FUNCTION OF PEX1

Crucial for peroxisome assembly

Cleaves ATP to release energy which then allows the PEX1
protein to move

Without this movement, PEX1 cannot pull PEX5 out of the
membrane, therefore arresting peroxisome import
THE MOVEMENT OF PEX1: AAA ATPASE
Rouiller et al., 2002
THE PEX1-G843D ALLELE


20-30% of all alleles resulting in Zellweger Spectrum
Disorder
Presents milder phenotype

No developmental defects, but multi-systemic effects due
to progressive peroxisome dysfunction over time
THE PEX1-G843D ALLELE

Single amino acid substitution: Glycine

Results in protein misfolding and degradation


Hindered ability to cleave ATP
Why do we see some function?


Still able to complex with PEX6 at membrane
Form a complex with reduced function
Aspartate
THE EFFECT OF PEX1 MISFOLDING
Fujiki et al.,2012
IMPROVING THE FUNCTION OF PEX1G843D

If protein is misfolded, can that be corrected?

In vitro studies using patient cell lines indicate:

PEX1-G843D may be amenable to proper folding
Temperature (30ºC), chemical + pharmacologic chaperones
 Peroxisome function can improve

IMPROVING THE FUNCTION OF PEX1G843D

> 2000 compounds screened


Flavonoids identified as effective in improving
peroxisomal import
Diosmetin
Works in presence of PEX1-G843D protein,
 Does not work in PEX1 null cells, PEX6 null cells, or
PEX 12 cells with a missense mutation
 Indicates some specificity, as expected with a
pharmacologic chaperone

DIOSMETIN: MECHANISM OF ACTION

Flavonoids observed to bind at ATP binding pocket
in other proteins
Nguyen et al. 2006
Diosmetin may bind at the ATP binding site of PEX1G843D.
DIOSMETIN: MECHANISM OF
ACTION
Flavonoids act on assembled membrane
complex
Gillian MacLean
observed that
diosmetin acts in
PEX1+PEX6 complex
at the peroxisomal
membrane
MacLean, 2013
DIOSMETIN: MECHANISM OF ACTION
Our hypothesis:
Following PEX1/PEX6 complex formation,
diosmetin binds at the ATP binding sites of PEX1G843D and improves its conformation.
Diosmetin binding is reversible.
Diosmetin is released, and the properly-folded
protein is now better able to bind ATP.
GOAL: DESIGNING A FLAVONOID THAT BEST
IMPROVES THE FUNCTION OF PEX1-G843D

STEPS:





Optimize drug structure of the for best fit into the
PEX1-G843D complex
Optimize drug structure to release a properly folded
PEX1-G843D
Ensure recovery of downstream peroxisome function
Minimize toxicity
Test in mouse model for clinical efficacy (Dr.
Steinberg)
OPTIMIZING DRUGS FOR PEX1-G843D:
PARTNERING WITH PHARMACEUTICAL
COMPANIES
Diosmetin, scbt.com

Pharmaceutical companies engineer many
elaborations of a core compound
Screen for best effect
 Minimal adverse effects

OPTIMIZING DRUGS FOR PEX1-G843D:
PARTNERING WITH PHARMACEUTICAL
COMPANIES
A not-for-profit organization that acts to bridge the gap
between basic research and later stage drug development.
OPTIMIZING DRUGS FOR PEX1-G843D:
PARTNERING WITH PHARMACEUTICAL
COMPANIES

In order to design the best chemical
substitutions, a company must know that the
drug acts on the target protein.
THE PATH FORWARD:
DEFINING HOW DIOSMETIN AND OTHER
PHARMACOLOGIC CHAPERONES WORK
Does it bind PEX1-G843D?

Require new tools

Develop artificial system to
model persoxisome
import/export

Develop best method to
over-express tagged PEX1G843D and PEX6

Develop a bioinformatic
approach to predict drug
docking
What is its effect on PEX1G843D?


ATPase activity/ ATP
hydrolysis/ ATP binding
Establish kinetics of binding
affinity
THE PATH FORWARD:
Chaperone therapy for other mutations

Potential of chemical chaperones to benefit a
broad ZSD patient population


Via nonspecific interaction with misfolded proteins
Develop general screening test to observe if lead
compounds could effectively treat other ZSD
mutations
THE PATH FORWARD:
Chaperone therapy for other mutations

BacMam assay



Baculovirus coupled with mammalian promoter
Easy expression of GFP-PTS1 reporter in any patient cell
line
Easy screen for drug response
Dolman et al., 2013
ACKNOWLEDGEMENTS
Nancy Braverman
Gillian MacLean
Panteha Saberian & Sara Birjandian
Steve Steinberg
Joe Hacia
NEOMED Institute
Patients and Families for kindly providing cells for
research
Funding Organizations: