Construction of a hybrid β-hexosaminidase subunit capable of forming stable homodimers that hydrolyze GM2 ganglioside in vivo.

Tay-Sachs or Sandhoff disease result from mutations in either the evolutionarily related HEXA or HEXB genes encoding respectively, the α- or β-subunits of β-hexosaminidase A (HexA). Of the three Hex isozymes, only HexA can interact with its cofactor, the GM2 activator protein (GM2AP), and hydrolyze GM2 ganglioside. A major impediment to establishing gene or enzyme replacement therapy based on HexA is the need to synthesize both subunits.

Pyrimethamine Derivatives: Insight into Binding Mechanism and Improved Enhancement of Mutant β-N-acetylhexosaminidase Activity.

In order to identify structural features of pyrimethamine (5-(4-chlorophenyl)-6-ethylpyrimidine-2,4-diamine) that contribute to its inhibitory activity (IC50 value) and chaperoning efficacy toward β-N-acetylhexosaminidase, derivatives of the compound were synthesized that differ at the positions bearing the amino, ethyl, and chloro groups. Whereas the amino groups proved to be critical to its inhibitory activity, a variety of substitutions at the chloro position only increased its IC50 by 2-3-fold. Replacing the ethyl group at the 6-position with butyl or methyl groups increased IC50 more than 10-fold.

Lysosomal abnormalities in hereditary spastic paraplegia types SPG15 and SPG11.

Objective: Hereditary spastic paraplegias (HSPs) are among the most genetically diverse inherited neurological disorders, with over 70 disease loci identified (SPG1-71) to date. SPG15 and SPG11 are clinically similar, autosomal recessive disorders characterized by progressive spastic paraplegia along with thin corpus callosum, white matter abnormalities, cognitive impairment, and ophthalmologic abnormalities. Furthermore, both have been linked to early-onset parkinsonism.

Synthesis of 1,5-dideoxy-1,5-iminoribitol C-glycosides through a nitrone-olefin cycloaddition domino strategy: identification of pharmacological chaperones of mutant human lysosomal β-galactosidase.

We report herein a newly developed domino reaction that facilitates the synthesis of new 1,5-dideoxy-1,5-iminoribitol iminosugar C-glycosides 7a-e and 8. The key intermediate in this approach is a six-membered cyclic sugar nitrone that is generated in situ and trapped by an alkene dipolarophile via a [2 + 3] cycloaddition reaction to give the corresponding isooxazolidines 10a-e in a "one-pot" protocol. The iminoribitol C-glycosides 7a-e and 8 were found to be modest β-galactosidase (bGal) inhibitors.

In cellulo examination of a beta-alpha hybrid construct of beta-hexosaminidase A subunits, reported to interact with the GM2 activator protein and hydrolyze GM2 ganglioside.

The hydrolysis in lysosomes of GM2 ganglioside to GM3 ganglioside requires the correct synthesis, intracellular assembly and transport of three separate gene products; i.e., the alpha and beta subunits of heterodimeric beta-hexosaminidase A, E.

Rapid assembly of a library of lipophilic iminosugars via the thiol-ene reaction yields promising pharmacological chaperones for the treatment of Gaucher disease.

A highly divergent route to lipophilic iminosugars that utilizes the thiol-ene reaction was developed to enable the rapid synthesis of a collection of 16 dideoxyiminoxylitols bearing various different lipophilic substituents. Enzyme kinetic analyses revealed that a number of these products are potent, low-nanomolar inhibitors of human glucocerebrosidase that stabilize the enzyme to thermal denaturation by up to 20 K. Cell based assays conducted on Gaucher disease patient derived fibroblasts demonstrated that administration of the compounds can increase lysosomal glucocerebrosidase activity levels by therapeutically relevant amounts, as much as 3.

A sensitive fluorescence-based assay for monitoring GM2 ganglioside hydrolysis in live patient cells and their lysates.

Enzyme enhancement therapy, utilizing small molecules as pharmacological chaperones, is an attractive approach for the treatment of lysosomal storage diseases that are associated with protein misfolding. However, pharmacological chaperones are also inhibitors of their target enzyme. Thus, a major concern with this approach is that, despite enhancing protein folding within, and intracellular transport of the functional mutant enzyme out of the endoplasmic reticulum, the chaperone will continue to inhibit the enzyme in the lysosome, preventing substrate clearance.

Oxidation of the primary hydroxyl group of galactose of galactaosyl ceramide analogue by chemical method-precursors for the synthesis of labeled conjugates.

Isotopic labeling of the C-6 of a model glycosphingolipid (2S, 3R, 4E)-2-(1-adamantanacetamido)-3-hydroxy-4-octadecenyl-beta-D-galactopyranoside, GalCAda, is described. Oxidation of (2S, 3R, 4E)-2-(1-adamantanacetamido)-3-(benzoyloxy)-4-octadecenyl-2,3,4-tri-O-benzoyl-beta-D-galactopyranoside with o-iodoxybenzoic acid gave the dialdoside derivative in good yield. Reduction of the dialdoside with sodium borodeuteride gave the deuterium labeled D-GalCAda, with a cumulative yield of 35%.