1. Challenges to therapy for peroxisome assembly disorders Nancy Braverman, MS, MD McGIll University-MCH-RI March 9 2010 HGEN 171-575

2. Properties of peroxisomes Spherical, single membrane bound, Diameter = 0.2 - 1 µm, several hundred/cell All eukaryotes

3. Peroxisomes originate from ER membranes and by fission of existing peroxisomes adapted from Annu Rev Genet. 2000;34:623-652. Sacksteder KA, Gould SJ. NEXT >> Click to view animation >>

4. 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.

5. Enzymatic pathways in peroxisomes Fatty acid oxidation (VLCFA, PA) H 2 O 2 detoxification (catalase) Docohexanoic acid (DHA) synthesis Bile acid synthesis Plasmalogen (ether phospholipid) synthesis Cholesterol synthesis Glyoxylate detoxification Lysine catabolism (pipecolic acid)

6. The 3 major metabolic pathways in peroxisomes

7. Properties of peroxisomal matrix proteins Contain Peroxisome Targeting Sequences (PTS) Imported as oligomers/fully assembled proteins Can have dual localizations in mitochondria, cytosol C - terminal (-SKL) Most matrix proteins Receptor is PEX5 -SKL PTS1PTS2 N - terminal (-R/KLX 5 Q/HL-) Presequence cleaved internally 3 enzymes only: Thiolase, PhyH, AGPS Receptor is PEX7 R/KLX 5 Q/HL -SKL

8. 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

9. 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

10. 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 A--------->B

11. 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?

12. PBD phenotypes correlate with biochemical severity

13. ControlZS PX # and size Matrix protein import IRD Phenotype correlates with severity of protein import defect, peroxisome number and size

14. PMP70 PTS1/PTS2 CATALASE 37 30 Mild PBD, PEX1-G843D/G843D

15. PEX1-G843D/ G843D, expressing GFP- PTS1 reporter No Treatment 30 o C Glycerol 5% 37 o C TMAO 200 uM 37 o C No Treatment 37 o C

17. Disorder-to-order conformational transitions in protein structure and its relationship to disease

18. Lower temperature Chaperone (protein or drug) – Nonspecific chemical chaperone – Pharmacologic chaperone Enzyme substrate Protein ligand (protein kinase and kinase inhibitor) Vitamin cofactor

20. 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

21. 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.

22. High throughput chemical screen Cells incubated in chemicals 2 days (2000 compounds) Negative control: media alone Positive control: TMAO and glycerol

23. 3 chemicals rescued import Data indicated that they can non- competitively bind to the ATP binding sites of proteins Potential pharmacologic chaperones!

24. 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

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

26. 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

27. Protein evolution depends on diet

28. Cell penetrating peptides: protein transduction therapy Endosomal exit to cytosol?

30. 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.

31. 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