Identification of Pharmacological Chaperones for Gaucher Disease and Characterization of Their Effects on β‐Glucocerebrosidase by Hydrogen/Deuterium Exchange Mass Spectrometry

Abstract

Point mutations in β-glucocerebrosidase (GCase) can result in a deficiency of both GCase activity and protein in lysosomes thereby causing Gaucher Disease (GD). Enzyme inhibitors such as isofagomine, acting as pharmacological chaperones (PCs), increase these levels by binding and stabilizing the native form of the enzyme in the endoplasmic reticulum (ER), and allow increased lysosomal transport of the enzyme. A high-throughput screen of the 50 000-compound Maybridge library identified two, non-carbohydrate-based inhibitory molecules, a 2,4-diamino-5-substituted quinazoline (IC50 5 μM) and a 5-substituted pyridinyl-2-furamide (IC50 8 μM). They raised the levels of functional GCase 1.5–2.5-fold in N370S or F213I GD fibroblasts. Immunofluorescence confirmed that treated GD fibroblasts had decreased levels of GCase in their ER and increased levels in lysosomes. Changes in protein dynamics, monitored by hydrogen/deuterium-exchange mass spectrometry, identified a domain III active-site loop (residues 243–249) as being significantly stabilized upon binding of isofagomine or either of these two new compounds; this suggests a common mechanism for PC enhancement of intracellular transport.

Diltiazem, a L-type Ca2+ channel blocker, also acts as a pharmacological chaperone in Gaucher patient cells

“…However, like IFG, Diltiazem did exhibit both of these properties at pH 6.5-7. We conclude that the Gcc enhancement mechanism of Diltiazem is the same as that of IFG or either of the other two non-carbohydrate based compounds we have identified by screening of a small molecule library [31,36], i.e., Diltiazem is a PC for Gcc in GD cells. Furthermore Diltiazem represents a PC with a non-carbohydrate framework that has already been evaluated in humans.…”

“…Primary/secondary antibodies and nuclear staining were as previously reported [36]. The primary Gcc antibody used was a rabbit polyclonal generated in our laboratory, and as judged by Western blots, cross-reacts with all the mutant forms of Gcc present in the cell lines analyzed in this report (data not shown).…”

“…5). However, Diltiazem contains a primary amine and two compounds we recently identified by high throughput screening of the Maybridge library as PCs for Gcc, 5-[((4-methylphenyl)thio]quina-zoline 2,4-diamine) (IC50 ~8 μM at pH 5.5) and 5-(3,5-dichlorophenoxy)-N-(4-pyridinyl)-2-furamide (IC50 ~5 μM at pH 5.5) [36], also contain either a primary or a secondary amine as part of their structure, and inhibit Gcc best at neutral pH [36]. Interestingly, IFG contains a secondary amine and similarly has been reported to inhibit Gcc best at neutral pH [33].…”

“…Diltiazem treatment also did not result in any further enhancement of activities in the GD or ATSD lines when co-administered with known PCs; i.e., isofagomine (IFG) [33], GD; pyrimethamine (PYR) [35], ATSD. The close similarity of Diltiazem, a benzothiazepine, to a Gcc inhibitor we recently identified with a benzodiazepine backbone ( [36], see Supplemental Table 1, compound BTB 03346) suggested that it may also act as a PC. Reexamination of Diltiazem as a potential PC for GD revealed that, while it does not express the biochemical characteristics of a PC at pH 4.5, these characteristics become evident at the neutral pH found in the ER.…”

2-Acetamino-1,2-dideoxynojirimycin—lysine hybrids as hexosaminidase inhibitors

“…23 These results suggest that the structural framework of compound 6a could be exploited for the design of additional efficacious pharmacological chaperones for GM2 gangliosidoses with low toxicity.…”