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ML009 : GCB (Glucocerebrosidase) Inhibitor

ML009

ML009

Target Name

Glucocerebrosidase

Target Alias

GCB

Target Class

Hydrolase

Mechanism of Action

Inhibitor of GCB

Biological / Disease Relevance

Gaucher disease

In Vitro Activity
IC50
0.074 uM
In Vitro Activity
Ki
0.052 uM
Inactive Control
Available

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

ML009

Sulfonamide analogue, 2

Physical & chemical properties
Molecular Weight 388.5 g/mol
Molecular Formula C17H16N4O3S2
cLogP 3.4
PSA 101
Storage -20
Solubility Up to 10mM in DMSO
CAS Number

SMILES:
CCC1=NN=C(S1)NC(C2=CC=C(NS(=O)(C3=CC=CC=C3)=O)C=C2)=O

InChI:
InChI=1S/C17H16N4O3S2/c1-2-15-19-20-17(25-15)18-16(22)12-8-10-13(11-9-12)21-26(23,24)14-6-4-3-5-7-14/h3-11,21H,2H2,1H3,(H,18,20,22)

InChIKey:
YMGXUJCEBOCIIT-UHFFFAOYSA-N

Activity

Summary activity statement /

ML009 (CID 2210290, SID 50105141, NCGC 00060210) is observed to be a potent and selective inhibitor of Glucocerebrosidase (GC) Chemotype 2 with an IC50 of 0.074 uM. The selectivity of probe ML009 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. ML009 is observed to be inactive against these 3 bioassays at concentrations up to 77 uM demonstrating high selectivity to GC. ML009 has been screened against 33 PubChem bioassays and is observed to be active only in the Glucocerebrosidase inhibition assay with an activity value of 0.153 uM.

In vitro assay - Selectivity

ML009 iminosugar nonyl-DNJ

glucocerebrosidase

 0.074 uM 0.103 uM

alpha-glucosidase

 Inactive 0.050 uM

alpha-galactosidase

 Inactive Inactive

beta-hexosaminidase

 Inactive Inactive

Summary /

Comparative data showing probe specificity for target. The selectivity of ML009 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 probe ML009 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) assay

ML009 N370S WT (control)

4.4 uM

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

13.3 uM

 20% increase in GC activity 0% increase in GC activity

40 uM

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

Summary /

Increase of glucocerebrosidase activity in Gaucher fibroblasts: The primary cells from Gaucher patients with N370S mutations (DMN 87.30) were treated with 4.4, 13.3 and 40 uM of SID 4264637 for 2 days in comparison with the wt. cells (GM5659). A treatment of 40uM inhibitors days resulted in a 40-90% 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.

In vitro assay - Mechanism of Action

Summary /

Mechanism of inhibition of probe: The effect of these GC inhibitor SID 4264637 on enzyme kinetics was studied in the enzyme assay to identify the mechanism of inhibition. SID 4264637 exhibited mixed inhibition.

References

  1. Identification of Modulators of the N370S Mutant Form of Glucocerebrosidase as a Potential Therapy for Gaucher Disease - Chemotype 2.
  2. Discovery, SAR, and Biological evaluation of Non-Inhibitory Small Molecule Chaperones of Glucocerebrosidase
  3. Vellodi, A. Lysosomal storage disorders. Br J Haematol 128, 413-431 (2005)
  4. 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
  5. 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)