Imino analogues of galactose: Inhibitors of human acid β-D-Galactosidase and putative pharmacological chaperones for the treatment of GM1-Gangliosidosis

GM1-gangliosidosis (GM1) and Morquio B disease (MBD) both evolve from mutations in the gene (GLB1) coding for human acid β-galactosidase (β-gal; EC 3.2.1.23). While in MBD the mutant enzyme is poorly active, but properly localized in the lysosomes, precursors may be prematurely degraded in many cases of GM1. Addition of competitive inhibitors, like imino analogues of galactose, to GM1 fibroblasts were shown to increase the residual β-gal activities, presumably due to normalized intracellular stability and localization. Therapeutic efforts for chronic GM1 have been based on this finding.

Twelve novel lipophilic and/or ionisable N-modified 1-deoxynojirimycin derivatives were synthesized and characterized as inhibitors of normal fibroblast β-gal activity via IC50- and Ki-values as well as for their potency to increase β-gal activity of GM1 and MBD fibroblasts in vivo. Most of the tested substances were efficient in vitro inhibitors (Ki <1µM; IC50 1–25µM) and eight of ten compounds showed an up to 14-fold increase of β-gal activity in proven chaperone-sensitive mutants (R201C; juvenile GM1) in vivo. Heterozygous cells carrying R201H beside other alleles (MBD; R201H/H281Y, juvenile GM1; R201H/R457X, juvenile GM1) also strongly responded to chaperones while cells from homozygous cases with MDB (W273L), infantile (Y333H; P549L; Y270D; A301V) or adult GM1 phenotypes (G438E) did not respond.

Thus the therapeutic benefit of chaperones depends on the specific effect of each mutation on the stability and localization of the resulting gene product. Codons 273 and 333, associated with MBD and juvenile GM1, respectively, are located within the catalytic site of the β-gal. While for W273L, normal amounts of mature β-gal have been found, little is known for other alleles. Current experiments characterize the gene products resulting from GLB1 mutations without or under the influence of potential chaperones with respect to subcellular localization, transport and catalytic properties of β-galactosidase. This may lead to improved mutation-specific design of therapeutic chaperones.