Transcript Challenges to therapy for peroxisome assembly disorders

Challenges to therapy for
peroxisome assembly disorders
Nancy Braverman, MS, MD
McGIll University-MCH-RI
March 9 2010
HGEN 171-575
Properties of peroxisomes
• Spherical, single membrane bound,
• Diameter = 0.2 - 1 µm, several hundred/cell
• All eukaryotes
Peroxisomes originate from ER membranes
and by fission of existing peroxisomes
Click to view animation >>
adapted from Annu Rev Genet. 2000;34:623-652.
Sacksteder KA, Gould SJ.
NEXT >>
Role of peroxins in matrix protein import
Click to view animation >>
Gould, Raymond, Valle.In: Metab & Molec Basis of Inh Dis. Ch 129 p. 3190.
Enzymatic pathways in peroxisomes
•
•
•
•
•
•
•
•
Fatty acid oxidation (VLCFA, PA)
H2O2 detoxification (catalase)
Docohexanoic acid (DHA) synthesis
Bile acid synthesis
Plasmalogen (ether phospholipid) synthesis
Cholesterol synthesis
Glyoxylate detoxification
Lysine catabolism (pipecolic acid)
The 3 major metabolic pathways in
peroxisomes
Properties of peroxisomal matrix
proteins
• Contain Peroxisome Targeting Sequences (PTS)
PTS1
PTS2
-SKL -SKL
C - terminal (-SKL)
Most matrix proteins
Receptor is PEX5
R/KLX5Q/HL
N - terminal (-R/KLX5 Q/HL-)
Presequence cleaved internally
3 enzymes only: Thiolase, PhyH, AGPS
Receptor is PEX7
• Imported as oligomers/fully assembled proteins
• Can have dual localizations in mitochondria,
cytosol
Genetic disorders of peroxisomes
• Multiple enzyme deficiencies: Peroxisomal
Biogenesis Disorders (PBD)
– Zellweger spectrum disorder (ZSD) (~1/60,000)
– Rhizomelic chondrodysplasia punctata spectrum
(RCDP)(~1/100,000)
• Single enzyme deficiencies
–
–
–
–
X-linked adrenoleukodystrophy (X-ALD) (~1/20,000)
3-methyl-CoA racemase deficiency
Adult Refsum disease
Hyperoxaluria Type I
Some single enzyme deficiencies can
mimic PBDs
• VLCFA oxidation → Zellweger spectrum
disorder
– Acyl-CoA oxidase
– D-Bifunctional protein (hydratase/dehydrogenase)
• Plasmalogen biosynthesis → RCDP spectrum
– DHAPAT (RCDP2)
– ADHAPS (RCDP3)
• Some PBDs mimic SEDs →
– Adult Refsum disease causes PEX7 deficiency
Develop therapies targeted
to the metabolic defects
A--------->B
•
•
Phytanic acid restriction
Reduction in VLCFA
dietary reduction
enhance VLCFA omega oxidation
reduce VLCFA synthesis
•
Supplementation with DHA, bile acids,
plasmalogens
Develop therapies targeted
to the molecular defects
• Enhance activity of a defective PEX proteinimprove protein folding
• Bypass the need for a specific PEX proteinupregulate a partner PEX protein
•
•
•
•
•
Induce peroxisome proliferation
Enzyme/PEX protein replacement therapy ?
Liver/stem cell transplant ?
Gene therapy ?
Manipulate the intestinal microbiome?
PBD phenotypes correlate
with biochemical severity
Phenotype correlates with severity of protein
import defect, peroxisome number and size
PX #
and
size
Matrix
protein
import
Control
ZS
IRD
Mild PBD, PEX1-G843D/G843D
PMP70
37
30
PTS1/PTS2
CATALASE
PEX1-G843D/ G843D, expressing GFPPTS1 reporter
No
Treatment
37 oC
No
Treatment
TMAO
200 uM
Glycerol 5%
37 oC
30 oC
37 oC
Disorder-to-order conformational transitions in
protein structure and its relationship to disease
• Lower temperature
• Chaperone (protein or drug)
– Nonspecific chemical chaperone
– Pharmacologic chaperone
Enzyme substrate
Protein ligand (protein kinase and kinase inhibitor)
Vitamin cofactor
Conformational changes of p97 AAA ATPase
during its ATPase cycle
Bind and hydrolyze ATP generating chemical energy that is converted into motion of
the molecule.
Motion used to pull PEX5 out of the membrane for another round of import
Role of peroxins in matrix protein import
Click to view animation >>
Gould, Raymond, Valle.In: Metab & Molec Basis of Inh Dis. Ch 129 p. 3190.
High throughput chemical screen
• Cells incubated in chemicals 2 days (2000 compounds)
• Negative control: media alone
• Positive control: TMAO and glycerol
3 chemicals rescued import
• Data indicated that they can noncompetitively bind to the ATP binding sites of
proteins
• Potential pharmacologic chaperones!
Pipeline for new drugs 5-10 yrs…
•
•
•
•
•
•
•
•
•
•
Develop HT assays to screen chemical libraries for compounds that recover
target function
Best to start with ‘in vivo’ assay, several complementary assays
Confirm ‘hits’ from the screening assay
Study structure-function relationships to develop best ‘lead’ compounds
Evaluate mechanisms of recovery
Asses pharmacokinetics: half-life, metabolism, excretion, recovery in the
brain, toxicity, tissue pathology (rodents)
Assay efficacy: animal models
Approval of drug for clinical trials or off-use label
Ensure drug supply, design and approval of clinical trial
Funding for clinical trial
Intracellular distribution of AGT, a protein
with an N-terminal MTS & C-terminal PTS1
Primary hyperoxaluria type 1
• 15-20% European and North American
population has Pro11Leu missense
substitution
– Decreased AGT stability
– Decreased enzymatic activity
– Enhances effect of additional mutations that are
predicted to be innocuous in its absence
– Redirects AGT to mitochondria
• Gly170Arg folding delay promotes mitochondrial
import
Protein evolution depends on diet
Cell penetrating peptides: protein
transduction therapy
Endosomal exit to cytosol?
Role of peroxins in matrix protein import
Click to view animation >>
Gould, Raymond, Valle.In: Metab & Molec Basis of Inh Dis. Ch 129 p. 3190.
Understanding the pathophysiology
may reveal other targets for therapy
 Selective inactivation of PEX5 gene in neural cells
Pex5-loxP x Nestin-cre (neurons,
oligodendrocytes and astrocytes)
Pex5-loxP x CNPase-cre (oligodendrocyte)
 Abnormal compaction of myelin
 Axonal damage and transport defects
 Reactive astrocytosis and microgliosis
 CD8+ T helper cells and increased cytokines