Challenges to therapy for peroxisome assembly disorders

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Challenges to therapy for peroxisome assembly
disorders

• Nancy Braverman, MS, MD
• McGIll University-MCH-RI

March 9 2010 HGEN 171-575
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Properties of peroxisomes

• Spherical, single membrane bound,
• Diameter 0.2 - 1 µm, several hundred/cell
• All eukaryotes

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Peroxisomes originate from ER membranes and by
fission of existing peroxisomes
NEXT gtgt
Click to view animation gtgt
adapted from Annu Rev Genet. 200034623-652.
Sacksteder KA, Gould SJ.
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Role of peroxins in matrix protein import
Click to view animation gtgt
Gould, Raymond, Valle.In Metab Molec Basis of
Inh Dis. Ch 129 p. 3190.
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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)

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The 3 major metabolic pathways in peroxisomes
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Properties of peroxisomal matrix proteins

• Contain Peroxisome Targeting Sequences (PTS)
• Imported as oligomers/fully assembled proteins
• Can have dual localizations in mitochondria,
  cytosol

PTS1
PTS2
-SKL
R/KLX5Q/HL
-SKL
N - terminal (-R/KLX5 Q/HL-) Presequence
cleaved internally 3 enzymes only Thiolase,
PhyH, AGPS Receptor is PEX7
C - terminal (-SKL) Most matrix
proteins Receptor is PEX5
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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

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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

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Develop therapies targeted to the metabolic
defects

• Phytanic acid restriction
• Reduction in VLCFA
• dietary reduction
• enhance VLCFA omega oxidation
• reduce VLCFA synthesis
• Supplementation with DHA, bile acids,
  plasmalogens

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Develop therapies targeted to the molecular
defects

• Enhance activity of a defective PEX protein-
• improve protein folding
• Bypass the need for a specific PEX protein-
• upregulate a partner PEX protein
• Induce peroxisome proliferation
• Enzyme/PEX protein replacement therapy ?
• Liver/stem cell transplant ?
• Gene therapy ?
• Manipulate the intestinal microbiome?

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PBD phenotypes correlate with biochemical severity
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Phenotype correlates with severity of protein
import defect, peroxisome number and size
PX and size
Matrix protein import
Control
ZS
IRD
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Mild PBD, PEX1-G843D/G843D
PMP70
CATALASE
PTS1/PTS2
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PEX1-G843D/ G843D, expressing GFP-PTS1 reporter
No Treatment 30 oC
No Treatment 37 oC
Glycerol 5 37 oC
TMAO 200 uM 37 oC
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Disorder-to-order conformational transitions in
protein structure and its relationship to disease
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• Lower temperature
• Chaperone (protein or drug)
• Nonspecific chemical chaperone
• Pharmacologic chaperone
• Enzyme substrate
• Protein ligand (protein kinase and kinase
  inhibitor)
• Vitamin cofactor

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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
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Role of peroxins in matrix protein import
Click to view animation gtgt
Gould, Raymond, Valle.In Metab Molec Basis of
Inh Dis. Ch 129 p. 3190.
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High throughput chemical screen

• Cells incubated in chemicals 2 days (2000
  compounds)
• Negative control media alone
• Positive control TMAO and glycerol

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3 chemicals rescued import

• Data indicated that they can non-competitively
  bind to the ATP binding sites of proteins
• Potential pharmacologic chaperones!

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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

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Intracellular distribution of AGT, a protein with
an N-terminal MTS C-terminal PTS1
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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

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Protein evolution depends on diet
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Cell penetrating peptides protein transduction
therapy
Endosomal exit to cytosol?
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Role of peroxins in matrix protein import
Click to view animation gtgt
Gould, Raymond, Valle.In Metab Molec Basis of
Inh Dis. Ch 129 p. 3190.
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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

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