| Summary: | Tay Sachs and Gaucher diseases are lysosomal storage disorders that are
caused by deficiencies in the lysosomal enzymes β-hexosaminidase A and
glucocerebrosidase, respectively. The glycolipid substrates of deficient β-hexosaminidase
A and glucocerebrosidase accumulate in lysosomal neurons, resulting
in neurodegenerative disorders. The enzyme deficiencies are caused by heritable
amino acid point mutations that result in unstable and potentially misfolded enzymes.
The mutant enzymes, although catalytically active in many cases, are destroyed by
cellular quality control mechanisms in the endoplasmic reticulum (ER).
A potential therapy for adult chronic Tay Sachs disease and Gaucher disease is
the use of enzyme inhibitors as chemical chaperones. To act as a chemical chaperone
the enzyme-specific inhibitor must stabilize the folded conformation of the enzyme
and thus prevent enzyme destruction by cellular quality control mechanisms. The
enzyme/inhibitor complex can then be transferred from the E R to the lysosome where
the substrate concentration is significantly high that some enzyme activity is restored.
In the chronic adult disease forms, only 10 % of normal enzyme activity is required
for a patient to become asymptomatic.
A competitive inhibitor for β-hexosaminidase A, NAG-thiazoline, was
synthesized and shown to be a potent inhibitor o f β-hexosaminidase A and the related
enzyme β-hexosaminidase B, with Ki values of 270 nM and 190 nM , respectively.
Chemical denaturation studies showed that NAG-thiazoline stabilizes a bacterial
hexosaminidase from Streptomyces plicatus (Sp. Hex.) and human P-hexosaminidase
B against guanidine hydrochloride denaturation. Thermal denaturation studies
showed that NAG-thiazoline stabilizes Sp. Hex., β-hexosaminidase B, and β-
hexosaminidase A against thermal denaturation. These results provide a chemical
explanation for recent results from collaborator Dr. Mike Tropak (The Hospital for
Sick Children, Toronto), which showed that NAG-thiazoline increases Phexosaminidase
A activity in Tay Sachs fibroblasts.
A mechanism based inactivator for glucocerebrosidase, 2-deoxy-2-fluoro- β-Dglucopyranosyl
fluoride, was synthesized and shown to stabilize glucocerebrosidase
against guanidine hydrochloride denaturation. N-Octyl-l-epivalienamine was
synthesized and shown to be a potent competitive inhibitor o f glucocerebrosidase with a Ki value of 75 nM and to stabilize glucocerebrosidase against thermal denaturation.
These results show that 2-deoxy-2-fluoro- β-D-glucopyranosyl fluoride and N-octyl-l-epivalienamine
stabilize the folded conformation of glucocerebrosidase and may act
as chemical chaperones in therapies for Gaucher disease.
An additional study was performed in which the synthetic capacity of a
glycosynthase developed from a β-glucuronidase from Thermotoga maritima was
explored. Using α-D-glucopyranuronosyl fluoride or α-D-galactopyranuronosyl
fluoride as donors, and pNP-glucoside, pNP-xyloside, and pNP-cellobioside as
acceptors, the E476A β-glucuronidase mutant catalyzed the formation of six novel
oligosaccharides.
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