1. Roscoe Brady, M.D. Scientist Emeritus National Institutes of Health Bethesda, Maryland USA

2. CURRENT APPROACHES TO TREAT PATIENTS WITH HEREDITARY METABOLIC STORAGE DISORDERS 1. ENZYME REPLACEMENT THERAPY 2. SUBSTRATE DEPLETION 3. CHEMICAL CHAPERONES 4. GENE THERAPY

3. Can Additional Strategies Be Developed?

4. For many years it was believed that alterations of the normal sequence of amino acids caused a reduction in the ability of glucocerebrosidase to degrade glucocerebroside that accumulates in patients with Gaucher disease.

5. My colleagues and I discovered that the decrease of glucocerebrosidase activity is due, at least in part, to a reduction of the amount of this enzyme in the cells and tissues of patients with Gaucher disease. (Lu J, et al. Proc Natl Acad Sci USA 2010; 107: 21665)

6. Alterations of the amino acid sequence of glucocerebrosidase cause abnormal folding of the mutated enzymes within cells. Cells cannot tolerate mis-folded proteins. An enzyme complex called the proteaosome destroys mis-folded proteins.

7. We investigated proteasomal degradation of mutated glucocerebrosidase in skin fibroblasts derived from patients with Gaucher disease with the N370S and L444P mutations.

8. The quantity of glucocerebrosidase is reduced in cells of patients with Gaucher disease. % of normal Type 1 Adult GD (N370S/N370S) 43. Type 2 Acute neuronopathic GD (L444P/L444P) 11. Type 3 Chronic neuronopathic GD (L444P/L444P) 22.

9. These changes cause a comparable reduction of the catalytic activity of glucocerebrosidase in cells of patients with Gaucher disease % of normal Type 1 GD (N370S/N370S) 42. Type 2 GD (L444P/L444P) 10. Type 3 GD (L444P/L444P) 25.

10. Why does this happen?

11. Cellular strategy of protein quality control

12. Biosynthesis, Folding and Transport of Glucocerebrosidase to Lysosomes messenger RNA The protein encoded by this gene is a molecular chaperone that is a member of the chaperonin containing TCP1 complex (CCT), also known as the TCP1 ring complex (TRiC). This complex consists of two identical stacked rings, each containing eight different proteins. Unfolded polypeptides enter the central cavity of the complex and are folded in an ATP-dependent manner. The TCP1 ring complex consists of two identical stacked rings, each containing eight different proteins. Unfolded polypeptides enter the central cavity of the complex and are folded in an ATP-dependent manner. Hsp70 prevents nascent peptides from aggregating and being rendered nonfunctional

13. Biosynthesis, Mis-folding and Degradation of Mutated Glucocerebrosidase Hsp90 is essential for the creation, maintenance and destruction of proteins. Its normal function is critical to maintaining the health of cells messenger RNA

14. Quantification of the increase in binding of N370S and L444P glucocerebrosidase mutants to the Hsp90 complex that facilitates degradation. Quantification of the reduction of binding of N370S and L444P glucocerebrosidase mutants to Hsp70 and TCP1 required for correct folding of nascent proteins.

15. The catalytic activity of the residual glucocerebrosidase in fibroblasts from patients with the N370S and L444P mutations was found to be normal.

16. Conclusions 1. Missense mutations destabilize glucocerebrosidase 2. Mutated glucocerebrosidase is partially degraded 3. Lysosomal localization is reduced 4. Rather than a decrease of intrinsic catalytic activity, proteasomal degradation underlies the reduction of enzymatic activity in patients with Gaucher disease caused by missense mutations

17. Would suppression of proteasomal degradation of mutated glucocerebrosidase provide an additional treatment option for patients with Gaucher disease?

18. Can proteostasis regulators promote the binding of N370S and L444P mutated glucoerebrosidases to chaperonins and reduce their degradation?

19. Histones Alkaline proteins in cell nuclei that package DNA into structural units

20. Histone deacetylases Enzymes that remove acetic acid residues from histones

21. Histone deacetylase inhibitors Decrease proteoasomal degradation thereby restoring the function of mis-folded proteins

22. Histone deacetylase inhibitors (HDACi) have been found to correct aberrant protein folding in: 1. Type 2 diabetes 2. Cystic fibrosis 3. Fibroblasts derived from patients with Type C Niemann-Pick disease

23. Can histone deacetylase inhibitors (HDACi) reduce proteasomal degradation of mutated enzymes in hereditary enzyme deficiency disorders such as Gaucher disease?

24. My colleagues and I discovered that histone deacetylase inhibitors prevent the degradation and restore glucocerebrosidase activity in Gaucher disease. (Lu J et al. Proc Natl Acad Sci USA 2011;108:21200)

25. Improved lysosomal trafficking of mutated glucocerebrosidase by HDACi

26. Suberoylanilide Hydroxamic acid (SAHA) o H o | H N I I Histone deacetylase inhibitor approved for the treatment of patients with cutaneous T cell lymphoma

27. Longer lasting histone deacetylase inhibitor Lixte Biotechnology, East Setauket, NY LB-205

28. Effect of histone deacetylase inhibitors on the quantity of glucocerebrosidase ( Percent of Normal ) N370S/N370S 38 68 81 L444P/L444P 12 43 41 Mutation Untreated Treated with SAHA Treated with LB-205

29. Effect of histone deacetylase inhibitors on the catalytic activity of mutated glucocerebrosidase ( Percent of Normal ) N370S/N370S 28 63 76 L444P/L444P 16 35 40 Mutation Untreated Treated with SAHA Treated with LB-205

30. Reported toxic effects of SAH A Nausea, vomiting, diarrhea, reduced appetite, headache, fatigue and indigestion that resolved during a week of rest. A safety and dose-response trial with LB-205 is under consideration

31. OVERVIEW Considerable efforts are underway concerning the development of innovative therapies for Gaucher disease and other hereditary metabolic disorders. The use of histone deacetylase inhibitors appears to be a promising therapeutic strategy for the following reasons: 1.They are administered orally thereby eliminating the necessity for intravenous infusions. 2.They cross the blood-brain barrier and are expected to be beneficial for patients with central nervous system involvement. 3.Because they are small molecules, it is presumed that their cost will be less than enzyme replacement and other therapies.

32. How do we proceed from here? 1.Determine the effect of HDAC inhibitors in murine models of Gaucher disease. 2.If positive results are obtained, carry out clinical studies in patients with Type 1 and neuronopathic forms of Gaucher disease.

33. Thank you