Pharmacological Chaperones for GCase that Switch Conformation with pH Enhance Enzyme Levels in Gaucher Animal Models

Abstract

Gaucher disease is a lysosomal storage disorder caused by mutations which destabilize the native folded form of GCase, triggering degradation and ultimately resulting in low enzyme activity. Pharmacological chaperones (PCs) which stabilize mutant GCase have been used to increase lysosomal activity through improving trafficking efficiency. By engineering their inherent basicity, we have synthesized PCs that change conformation between the ER and the lysosomal environment, thus weakening binding to GCase after its successful trafficking to the lysosome. NMR studies confirmed the conformational change while X‐ray data reveal bound conformations and binding modes. These results were further corroborated by cell studies showing increases in GCase activity when using the pH‐switchable probe at low dosing. Preliminary in vivo assays with humanized mouse models of Gaucher showed enhanced GCase activity levels in relevant tissues, including the brain, further supporting their potential.

Glycosidase-targeting small molecules for biological and therapeutic applications

“…29). 222 Iminosugar moieties have been employed extensively to design inhibitors of various glycosidases. However, these types of inhibitors tend to accumulate mainly in lysosomes with low pHs, owing to the presence of a basic nitrogen center.…”

From Sweet Molecular Giants to Square Sugars and Vice Versa

“…proper folding without interfering with GlcCer turnover may indeed lead to better restoration of the residual enzyme activity. 73 No doubt there was an element of risk in this project, for we had no information on the localization of the non-catalytic binding site, but this was somehow counterbalanced by the design of the Janus-type inhibitors combining two enantiomeric heads. Such a structural feature facilitated the synthesis of the heterodimeric inhibi-tors and, thus, their rapid evaluation as GCase PCs.…”

Mechanistic Insights into Dibasic Iminosugars as pH-Selective Pharmacological Chaperones to Stabilize Human α-Galactosidase

“…More recently, pharmacological chaperone therapy (PCT), the use of pharmacological chaperones (PCs) to either enhance the trafficking of endogenous mutant proteins to the lysosome and/or minimize the denaturation of exogenous recombinant enzymes in the circulatory system, has emerged as an alternative approach to treat FD and many other LSDs (Figure a). − For example, 1-deoxygalactonojirimycin (DGJ, 1 ) is the first PC approved by the U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) for treating patients with FD. , Most reported PCs such as the iminosugars DGJ and 1-deoxynojirimycin (DNJ, 2 ) are competitive inhibitors since PC development starting from glycosidase substrates or transition-state mimics is easier than starting from scratch (Figure b). − However, the inhibitory activity of these PCs can diminish their therapeutic benefit. Several strategies have been developed to reduce their inhibitory effects, such as the use of inhibitors that decompose in the acidic environment within lysosomes or transient inactivators that can be slowly hydrolyzed by their target enzymes. − A major challenge associated with the development of PCs to treat LSDs is the need for their binding to be pH-dependent; the PCs should preferentially bind to their targets in the ER (pH 7.1) and the circulatory systems (pH 7.4) rather than in lysosomes (pH 4.5–5.0), where substrate processing should not be inhibited.…”

“…Several strategies have been developed to reduce their inhibitory effects, such as the use of inhibitors that decompose in the acidic environment within lysosomes or transient inactivators that can be slowly hydrolyzed by their target enzymes. 20 − 22 A major challenge associated with the development of PCs to treat LSDs is the need for their binding to be pH-dependent; the PCs should preferentially bind to their targets in the ER (pH 7.1) and the circulatory systems (pH 7.4) rather than in lysosomes (pH 4.5–5.0), where substrate processing should not be inhibited.…”