Synthesis and testing of isofagomine and derivatives as inhibitors of human glucocerebrosidase

Gaucher disease is a lysosomal storage disorder in which the activity of the enzyme glucocerebrosidase (GCase) is deficient. As a result, the substrate of GCase, glucosylceramide, accumulates in lysosomes of Gaucher patients, causing enlargement of the spleen and liver, bone deformity and neurodegeneration. Deficient GCase activity results from point mutations in the gene encoding for GCase, which cause the protein to be misfolded. The misfolded mutant form of GCase, although catalytically competent, is identified and degraded by the endoplasmic reticulum-associated degradation (ERAD) pathway; thereby never reaching the lysosome or its substrate, resulting in accumulation. A threshold GCase activity level of 11-15% is all that is needed in order to be asymptomatic, with most Gaucher patients retaining between 5-20% of GCase activity levels. Therefore, therapeutic treatment of Gaucher disease only needs to impart modest increases in GCase activity in order to see large clinical improvements. An emerging therapy for Gaucher disease uses competitive enzyme inhibitors as pharmacological chaperones (PCs), which act to increase GCase levels in the lysosome. The PC binds and stabilizes the native, folded form of GCase, thereby allowing the enzyme to escape ERAD and resume proper trafficking to the lysosome. Once the mutant GCase/PC complex is delivered to the lysosome, the relatively high concentration of accumulated glucosylceramide will compete for the active site of GCase, thereby displacing the PC as normal glycolipid degradation activity resumes. Isofagomine (IFG), a known competitive inhibitor of GCase, was synthesized along with five other C6-alkyl IFG derivatives; of which four lEG derivatives were novel compounds. All six compounds were shown to be potent competitive inhibitors of GCase with measured K, values ranging from 0.2 nM to 610 nM. As a strategy to suppress any inhibitory effects of the PCs after GCase reaches the lysosome, a potentially pH-labile acetal moiety was incorporated into IFG. Unfortunately, the synthesized acetal is very stable and unlikely to cleave under physiological conditions. All molecules synthesized,have been, or will be, sent to our collaborators, Dr. Mike Tropak and Dr. Don Mahuran at the Hospital for Sick Children in Toronto for PC activity testing in Gaucher cell lines.

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