Lipid-mimicking phosphorus-based glycosidase inactivators as pharmacological chaperones for the treatment of Gaucher's disease

Abstract

Gaucher's disease, the most prevalent lysosomal storage disorder, is caused by missense mutation of the GBA gene, ultimately resulting in deficient GCase activity, hence the excessive build-up of cellular glucosylceramide. Among different therapeutic strategies, pharmacological chaperoning of mutant GCase represents an attractive approach that relies on small organic molecules acting as protein stabilizers. Herein, we expand upon a new class of transient GCase inactivators based on a reactive 2-deoxy-2-fluoro-β-d-glucoside tethered to an array of lipid-mimicking phosphorus-based aglycones, which not only improve the selectivity and inactivation efficiency, but also the stability of these compounds in aqueous media. This hypothesis was further validated with kinetic and cellular studies confirming restoration of catalytic activity in Gaucher cells after treatment with these pharmacological chaperones.

Fig. 1. (a) GCase double-displacement mechanism acting on its natural substrate or a 2FGlc-derived inactivator; (b) chemical structure of glucosylceramide, GCase natural substrate.

Scheme 1. Synthesis of 2-fluorosugars bearing phosphorus-based aglycones: (a) 11b: diethyl phosphite, n-octanol, NaH, 160 °C; (b) 11a, NaH, THF, alkyl halide, 12a: with n-butyl iodide, 12b: with BnBr; (c) 12c: 11b, Na, toluene, 1-bromooctane, reflux; (d) 12d: 11b, Cs₂CO₃, DMF, BnBr, TBAI; (e) 13a, 13b and 13d: TMSI, CH₂Cl₂; (f) 13c: NaI, pyridine, reflux; (g) 3,4,6-tri-O-acetyl-2-deoxy-2-fluoro-d-glucopyranosyl bromide, THF/MeCN/CH₂Cl₂ or MeCN, Ag₂CO₃16a: from 13a, 16b: from 13b, 16c: from 13c, 16d: from 13d; (h) 14b: n-octylamine, Et₃N, toluene; (i) 14c: n-octanol, quant.; (j) 15a: from 14a with n-octylmagnesium bromide, Et₂O/toluene (4 : 1); (k) Et₃N, toluene, corresponding amine 15b: POCl₃, n-octylamine, 15c: from 14b with aniline, 15d: from 14c with n-octylamine, 15e: from 14a with n-octylamine; 15f: from 14a with n-dodecylamine; (l) 3,4,6-tri-O-acetyl-2-deoxy-2-fluoro-d-glucopyranose, CH₂Cl₂, Et₃N, 3 Å molecular sieves, 16e: from 15a, 16f: from 15b, 16g: from 15c, 16h: from 15d, 16i: from 15e, 16j: from 15f; (m) Na, MeOH, 1: from 16a, 2: from 16b, 3: from 16c, 4: from 16d, 5: from 16e, 6: from 16f, 7: from 16g, 8: from 16h, 9: from 16i, 10: from 16j; compounds 11a and 14a are commercially available.

Fig. 2. Glucosylceramide-mimicking 2-deoxy-2-fluoroglucopyranosyl phosphonates, phosphoramidates and phosphordiamidates 1–10 prepared in this study.

Fig. 3. PAGE gels imaged with the fluorescent activity-based probe MDW933 (top) and Western blot (bottom), showing changes in active and total [GCase] over time (h) after treatment with phosphonate 4, respectively. Lane 0 represents untreated cells.