Pharmacological Enhancement of β-Hexosaminidase Activity in Fibroblasts from Adult Tay-Sachs and Sandhoff Patients

Abstract

Tay-Sachs and Sandhoff diseases are lysosomal storage disorders that result from an inherited deficiency of beta-hexosaminidase A (alphabeta). Whereas the acute forms are associated with a total absence of hexosaminidase A and early death, the chronic adult forms exist with activity and protein levels of approximately 5%, and unaffected individuals have been found with only 10% of normal levels. Surprisingly, almost all disease-associated missense mutations do not affect the active site of the enzyme but, rather, inhibit its ability to obtain and/or retain its native fold in the endoplasmic reticulum, resulting in its retention and accelerated degradation. By growing adult Tay-Sachs fibroblasts in culture medium containing known inhibitors of hexosaminidase we have raised the residual protein and activity levels of intralysosomal hexosaminidase A well above the critical 10% of normal levels. A similar effect was observed in fibroblasts from an adult Sandhoff patient. We propose that these hexosaminidase inhibitors function as pharmacological chaperones, enhancing the stability of the native conformation of the enzyme, increasing the amount of hexosaminidase A capable of exiting the endoplasmic reticulum for transport to the lysosome. Therefore, pharmacological chaperones could provide a novel approach to the treatment of adult Tay-Sachs and possibly Sandhoff diseases.

Treatment of Human Fibroblasts Carrying NPC1 Missense Mutations with MG132 Leads to an Improvement of Intracellular Cholesterol Trafficking

“…Protein misfolding has been implicated in many lysosomal storage disorders, such as Gaucher disease (Alfonso et al 2005), Fabry disease (Fan et al 1999), GM1 gangliosidosis (Tropak et al 2004), and Pompe disease (Parenti et al 2007). In this paper we demonstrated that, besides the p.I1061T NPC mutant protein, other 6 NPC1 mutant variants are unstable and subjected to an increased rate of proteasomal degradation.…”

Crystal Structure of β-Hexosaminidase B in Complex with Pyrimethamine, a Potential Pharmacological Chaperone

“…NGT, a sulfur analogue of the oxazolinium ion intermediate formed during substrate turnover, was found to be an excellent candidate with a K i of 300 nM. 11 Subsequent screens identified PYR as a potential PC having many favorable qualities such as its ability to cross the blood-brain barrier and the fact that it was already FDA approved, thereby eliminating the need for expensive preclinical trials. 13 The encouraging results from a small phase I/II clinical trial of PYR for late onset G M2 gangliosidosis have recently been reported.…”

“…10 Several molecules that act as inhibitors of HexA and HexB have been identified as potential pharmacological chaperones (PC) for late-onset G M2 gangliosidosis, including known inhibitors of Hex such as N-acetylglucosamine thiazoline (NGT). 11 Additionally, two libraries have been screened, the Maybridge library 12 (50 000 compounds) and the NINDs library 13 (1040 FDA approved compounds). These screens have led to the discovery that pyrimethamine (PYR, 5-(4-chlorophenyl)-6-ethyl-2,4-pyrimidinediamine) is also a good candidate for a PC.…”

2-Acetamino-1,2-dideoxynojirimycin—lysine hybrids as hexosaminidase inhibitors

“…Intracellular β-N-acetylhexosaminidase activity-Adult Tay-Sachs (homozygous for alpha subunit G269S mutation) and infantile Sandhoff (homozygous for the 16-kb HEXB deletion mutation) fibroblasts 11 were treated with an escalating dose of 6a (dissolved in DMSO) for 5 days. Intracellular β-N-acetylhexosaminidase A/S activity was measured using the fluorogenic substrate 4-methylumbelliferyl N-acetylglucosamine-6-sulfate as previously described.…”

“…10 More recently, hexosaminidase inhibitor 3 was examined as a pharmacological chaperone for lysosomal β-Nacetylhexosaminidase A/S (Hex A/S) in fibroblasts derived from patients with Sandhoff and Tay-Sachs diseases. 8,11 Following up on our observation that some fluorescently labelled derivatives of the Dglucosidase inhibitor 2,5-dideoxy-2,5-imino-D-mannitol (DMDP) are powerful inhibitors exceeding the parent compound's activity by two orders of magnitude, 12 we have reported the syntheses and glycosidase inhibitory activities of various pyranoid N-alkylated iminoalditols featuring fluorescent tags such as dansyl moieties attached to simple Nsubstituents. 13 Based on their encouraging inhibitory activities, we envisaged more convenient properties with compounds providing a suitably positioned amine for tagging as well as an additional 'handle' for chain extension and with a view to variation of the Nsubstituent's chemical as well as biochemical properties.…”