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ML008 : GCB (Glucocerebrosidase) Inhibitor

ML008

ML008

Target Name

Glucocerebrosidase

Target Alias

GCB

Target Class

Hydrolase

Mechanism of Action

Inhibitor of GCB

Biological / Disease Relevance

Gaucher disease

In vitro assay
IC50
0.035 uM
In vitro assay
Ki
0.037 uM

Target Information

Beta-glucocerebrosidase catalyzes the hydrolysis of beta-glucocerebroside to glucose and ceramide. The inherited deficiency of beta-glucocerebrosidase results in Gaucher disease, which is characterized by a wide variety of symptoms including hepatosplenomegaly, anemia, thrombocytopenia, bony lesions and bone marrow infiltration with characteristic storage cells, known as Gaucher cells. It is believed that improper folding and trafficking of beta-glucocerebrosidase may contribute to the phenotypes observed. It is suggested that the pharmacological chaperone stabilizes the glucocerebrosidase conformation to prevent misfolding and premature degradation, and helps its trafficking from the ER to its functional site, the lysosome. Therefore, a small molecule beta-glucocerebrosidase inhibitor used as a pharmacological chaperone offers a therapeutic alternative. Not only could it bind to the enzyme to stabilize the conformation and help to improve protein trafficking, but it could also be designed to cross the blood-brain barrier to be used as a potential therapy for neuronopathic Gaucher disease, where currently no efficacious therapy is available.

Properties

ML008

NCGC00092410

Physical & chemical properties
Molecular Weight 353.5 g/mol
Molecular Formula C12H27N3O2
cLogP 4.3
PSA 54.5
Storage
Solubility
CAS Number

SMILES:
CC1=CC(N2CCOCC2)=NC3=CC=C(C=C13)NC(C4CCCCC4)=O

InChI:
InChI=1S/C21H27N3O2/c1-15-13-20(24-9-11-26-12-10-24)23-19-8-7-17(14-18(15)19)22-21(25)16-5-3-2-4-6-16/h7-8,13-14,16H,2-6,9-12H2,1H3,(H,22,25)

InChIKey:
BXVNPOBNVRBOOV-UHFFFAOYSA-N

Activity

Summary activity statement /

ML008 (SID 26753329, CID 5067281, NCGC00092410) is observed to be a potent and selective inhibitor of Glucocerebrosidase (GC) Chemotype 1 with an IC50 of 35 nM. The selectivity of probe ML008 was measured in 3 other hydrolases including alpha-glucosidase, alpha-galactosidase, and beta-N-acetyldglucosaminidase (HEX). These enzymes are all lipid hydrolases and shared the same metabolic pathways as GC. ML008 is observed to be inactive against these 3 bioassays at concentrations up to 77 uM demonstrating high selectivity to GC. ML008 has been screened against 48 PubChem bioassays and is observed to be active in 10 assays; among which only the GC primary and confimatory qHTS showed AC50 in the nanomolar range.

In vitro assay - Selectivity

ML008 iminosugar nonyl-DNJ

glucocerebrosidase

 0.035 uM 0.103 uM

alpha-glucosidase

 Inactive 0.050 uM

alpha-galactosidase

 Inactive Inactive

beta-hexosaminidase

 Inactive Inactive

Summary /

The selectivity of ML008 was measured in three other hydrolases including α-glucosidase, α-galactosidase, and β-N-acetylglucosaminidase (β-N-acetylhexosaminidase, HEX). Deficiencies in α-glucosidase, α-galactosidase and β-hexosaminidase result in Pompe disease, Fabry disease and Tay-Sachs or Sandhoff disease, respectively, all genetic disorders of lysosomal lipid metabolism similar to Gaucher disease (Vellodi A, 2005). Substrates of these three enzymes labeled with the blue fluorophore 4-methylumbelliferone were used with a GC enzyme assay using the substrate 4-methylumbelliferyl-β-D-glucopyranoside (4MU-β−Glc) as control. These four enzyme assays were performed in parallel with the 1536-well plates (Urban DJ, 2008). Results showed that SID 26753329 did not inhibit the activities of α-glucosidase, α-galactosidase, or β-hexosaminidase at concentrations up to 77 μM, demonstrating high selectivity to GC. In contrast, the iminosugar nonyl-DNJ was found to inhibit both GC and α-glucosidase, with IC50 values of 0.103 and 0.050 μM, respectively. The IC50 values of the compounds in the GC enzyme assay using the blue fluorogenic substrate 4MU-β−Glc were like those using fluorogenic substrate Res-β-Glc.

Cellular Activity - Primary Cell (Gaucher fibroblast) activity

ML008 N370S mutation WT (Control)

4.4 uM

 20% increase in GC activity 20% increase in GC activity

13.3 uM

 20% increase in GC activity 5% increase in GC activity

40 uM

 90% increase in GC activity 20% increase in GC activity

Summary /

Increase of glucocerebrosidase activity in Gaucher fibroblasts: Primary cells from Gaucher patients with N370S mutations (fibroblast lines DMN 87.30; genotype N370S/N370S) were treated with 4.4, 13.3 and 40 uM of ML008 (SID 26753329), for 2 days in comparison with the GM5659 WT cells (control). A treatment of 40 uM inhibitor resulted in a 90% increase of the mutant enzyme (N370S) activity in fibroblasts from Gaucher patients while the increase of normal enzyme activity was much smaller. This result indicates that these inhibitors may stabilize the mutant enzyme protein, help its proper folding/trafficking and thus increase its activity in the cell-based assay. Data represent three independent experiments performed with three replicates per sample.

Cellular Activity - Immunofluorescence staining: GC trafficking

Summary /

Increase of lysosomal glucocerebrosidase amount in Gaucher fibroblasts: The effects of ML008 (compound 1), SID 847960 (compound 3), an inactive aminoquinoline analog (compound 22), and the known iminosugar inhibitor nonyl-DNJ (compound 5) on the mutant GC trafficking were studied in fibroblast cell lines from patients with genotype N370S/N370S. A wild-type cell line was used as a control. Dual labeling with polyclonal GC antibody and a lysosomal marker revealed that GC was localized to the lysosomes in the wild-type cells, whereas only a limited amount of enzyme reached the lysosome in a N370S mutant line in the absence of compounds (Figure 1A). Treatment of two different N370S mutant fibroblasts with ML008 (compound 1), and, to a lesser extent, SID 847960 (compound 3), resulted in increased co-localization (an increase of GC protein in lysosome), suggesting improvement of GC trafficking (Figure 1B). This change was not seen in mutant cells treated with DMSO alone, compound 22, or with the known inhibitor nonyl-DNJ.

 

Figure 6: (A) Dual labeling with polyclonal GC antibody (GC Ab, red) and a lysosomal marker (LysoTracker, green) in untreated WT (GM 3348) and N370S (DMN 87.30) fibroblasts and in the N370S line treated with 40 uM ML008 (compound 1). Overlay images demonstrating colocalization (yellow) of GC Ab with the lysosomal marker indicate potential improvement in GC trafficking. (B) Immunofluorescence staining of two N370S mutant fibroblast lines, DMN 83.137 and DMN 87.30, grown with 40 uM compounds. Cells were co-stained with GC Ab and LysoTracker. Overlay images are shown for cells treated with DMSO (control), 5 (nonyl-DNJ), active compounds 1, 2, and 3, and inactive analog 22. Although there is some lysosomal colocalization in both cell lines, compound 1 and, to a lesser extent, compound 3, which significantly increased GC activity in the cell-based assay, show increased yellow fluorescence, demonstrating an increase of GC in the lysosomal compartment.

In vitro activity - Mechanism of inhibition

Summary /

Mechanism of inhibition of the probe: The effect of GC inhibitor SID 26753329 on enzyme kinetics was studied in the enzyme assay to identify the mechanism of inhibition. SID 26753329 exhibited mixed inhibition.

References

  1. Vellodi, A. Lysosomal storage disorders. Br J Haematol 128, 413-431 (2005).
  2. Urban, DJ et al. Development and Validation of Two Miniaturized Glucocerebrosidase Enzyme Assays for High-Throughput Screening. Combi Chem High Thr Screen. Dec;11(10):817-24. 2008
  3. Zheng et al. Three classes of glucocerebrosidase inhibitors identified by quantitative high-throughput screening are chaperone leads for Gaucher disease. PNAS 104(32): 13192–13197 (2007)