roscoe brady, m.d. scientist emeritus national institutes of health bethesda, maryland usa
TRANSCRIPT
CURRENT APPROACHES TO TREAT PATIENTS WITH
HEREDITARY METABOLIC STORAGE DISORDERS
1. ENZYME REPLACEMENT THERAPY
2. SUBSTRATE DEPLETION
3. CHEMICAL CHAPERONES
4. GENE THERAPY
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.
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 enzymein the cells and tissues of patients with Gaucher disease.
(Lu J, et al. Proc Natl Acad Sci USA 2010; 107: 21665)
Alterations of the amino acid sequence ofglucocerebrosidase 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.
We investigated proteasomal degradation of mutated glucocerebrosidase in skin fibroblasts derived from patients with Gaucher disease with the N370S and L444P mutations.
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.
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.
Biosynthesis, Folding and Transport of Glucocerebrosidase to Lysosomes
messenger RNA
The TCP1 ring complex consists of two identical stacked rings, eachcontaining eight different proteins. Unfolded polypeptides enter thecentral cavity of the complex and are folded in an ATP-dependent manner.
Hsp70 prevents nascent peptides from aggregating and being rendered nonfunctional
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
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.
The catalytic activity of the residual glucocerebrosidase in fibroblasts from patients with the N370S and L444P mutations was found to be normal.
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
Would suppression of proteasomal degradation of mutated glucocerebrosidase provide an additional treatment option for patients with Gaucher disease?
Can proteostasis regulators promote the binding of N370Sand L444P mutated glucoerebrosidases to chaperoninsand reduce their degradation?
Histone deacetylase inhibitors
Decrease proteoasomal degradation thereby restoring the function of mis-folded proteins
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
Can histone deacetylase inhibitors (HDACi) reduce proteasomal
degradation of mutated enzymes in hereditary enzyme deficiency disorders such as Gaucher disease?
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)
Suberoylanilide Hydroxamic acid (SAHA)
oH
o
|
H
N
I
I
Histone deacetylase inhibitor approved for the treatment of patients with cutaneous T cell lymphoma
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
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
Reported toxic effects of SAHA
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
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.
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.