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Discovery, SAR, and Biological Evaluation of Non-inhibitory Chaperones of Glucocerebrosidase

, , , , , , , , , , , , and .

Author Information

,a ,b ,a ,b ,c ,c ,b ,c ,c ,c ,b ,b,* and a,*.

a Specialized Chemistry Center, The University of Kansas, 2034 Becker Drive, Lawrence, KS 66047.
b NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, 9800 Medical Center Drive, Rockville, MD, 20850.
c Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, 20892; USA.
* To whom correspondence should be addressed: Email: ude.uk@ebuaj and ; vog.hin.liam@jnaguram

Received: ; Last Update: March 7, 2013.

Gaucher disease is a rare genetic lysosomal storage disease characterized by a loss of function in the glucocerebrosidase (GCase) enzyme, which is responsible for hydrolyzing glucocerebroside (GC) in the lysosome. When cells die, macrophages use GCase to break down GC, a major constituent of cell walls. With deficient functional GCase, GC accumulates within the lysosome, giving rise to the appearance of bloated Gaucher cells; this is a hallmark of the disease. Certain mutated GCase proteins, after production in the endoplasmic reticulum (ER), do not fold properly and are degraded via the proteasome pathway instead of being transported to the lysosome. One therapeutic strategy is to develop small molecule chaperones, which upon binding to GCase ensure proper folding and subsequent transport of the mutant protein to the lysosome, where it can resume activity. The main challenge in the development of molecular chaperones for Gaucher disease is that all of the previously described chaperones are inhibitors of the enzyme. This complicates their clinical development, because it is difficult to generate an appropriate in vivo exposure at which a compound exhibits chaperone activity, but does not inhibit the enzyme’s function. Using high throughput screening, we have identified two chemical series that do not inhibit the enzyme’s action, but can still facilitate its translocation to the lysosome as measured by immunostaining of glucocerebrosidase in patient fibroblasts. These chemical series are exemplified by ML198 and ML266. These compounds serve as starting points to develop a novel approach towards small molecule treatment for patients suffering from Gaucher disease.

Assigned Assay Grant #: R03MH086442

Screening Center Name & PI: NIH Chemical Genomics Center, Christopher P. Austin

Chemistry Center Name & PI: NIH Chemical Genomics Center, Christopher P. Austin

Chemistry Center Name & PI: The University of Kansas Specialized Chemistry Center, Jeffrey Aubé

Assay Submitter & Institution: Ellen Sidransky, NHGRI

PubChem Summary Bioassay Identifier (AID): 2593

Probe Structure & Characteristics

Image ml266fu1
CID/ML#Target NameIC50/EC50 (μM) [SID, AID]Anti-target Name(s)IC50/EC50 (μM) [SID, AID]Fold SelectiveSecondary Assay(s) Name: IC50/EC50 (nM) [SID, AID]
CID 46907762/ML198N370S GCase from Tissue Homogenate0.4 μM [SID 99368009, AID 488854]Acid alpha-glucosidase>57 μM [SID 99368009, AID 2577]> 100 foldChaperone Activity in N370S GCase fibroblasts at 1 μM [SID 99368009, AID 588853]
CID 46943215/ML266N370S GCase from Tissue Homogenate2.5 μM [SID 46943215, AID 2590]Acid alpha-glucosidase>57 μM [SID 46943215, AID 2577]> 22 foldChaperone Activity in N370S GCase fibroblasts at 1 μM [SID 46943215, AID 588853]

1. Recommendations for Scientific Use of the Probe

The class of pyrazolopyrimidines and salicylic acid derivatives disclosed here function as activators of GCase (glucocerebrosidase or acid beta glucosidase) in a functional N370S (the most common mutation in Gaucher disease) spleen homogenate assay, and show chaperone activity in a translocation assay using patient-derived fibroblasts. These compounds, due to the lack of their inhibitory activity, do not have the risk of inhibiting enzyme function after transport to the lysosome. Furthermore, unlike previous iminosugar chaperones that may have had side effects because of a lack of selectivity against other glucosidases, these compounds are also quite selective for GCase activity. Thus, these first-in-class non-inhibitor chaperones can be used as tools to further evaluate their therapeutic potential in various models of Gaucher disease. These compounds also demonstrate a lack of activity towards substrate hydrolysis of the structurally related acid alpha glucosidase and alpha galactosidase, thus appear to be selective towards GCase.

2. Materials and Methods

ML198: All air or moisture sensitive reactions were performed under positive pressure of nitrogen with oven-dried glassware. Anhydrous solvents such as dichloromethane, N,N-dimethylformamide(DMF), acetonitrile, methanol and triethylamine were obtained by purchasing from Sigma-Aldrich. Preparative purification was performed on a Waters semi-preparative HPLC. The column used was a Phenomenex Luna C18 (5 micron, 30 × 75 mm) at a flow rate of 45 mL/min. The mobile phase consisted of acetonitrile and water (each containing 0.1% trifluoroacetic acid). A gradient of 10% to 50% acetonitrile over 8 minutes was used during the purification. Fraction collection was triggered by UV detection (220 nM). Analytical analysis was performed on an Agilent LC/MS (Agilent Technologies, Santa Clara, CA). Method 1: A 7 minute gradient of 4% to 100% Acetonitrile (containing 0.025% trifluoroacetic acid) in water (containing 0.05% trifluoroacetic acid) was used with an 8 minute run time at a flow rate of 1 mL/min. A Phenomenex Luna C18 column (3 micron, 3 × 75 mm) was used at a temperature of 50°C. Method 2: A 3 minute gradient of 4% to 100% Acetonitrile (containing 0.025% trifluoroacetic acid) in water (containing 0.05% trifluoroacetic acid) was used with a 4.5 minute run time at a flow rate of 1 mL/min. A Phenomenex Gemini Phenyl column (3 micron, 3 × 100 mm) was used at a temperature of 50 °C. Purity determination was performed using an Agilent Diode Array Detector on both Method 1 and Method 2. Mass determination was performed using an Agilent 6130 mass spectrometer with electrospray ionization in the positive mode. 1H NMR spectra were recorded on Varian 400 MHz spectrometers. Chemical Shifts are reported in ppm with undeuterated solvent (DMSO-H6 at 2.50 ppm) as the reference for DMSO-d6 solutions. All analogs for assays have purity greater than 95% based on both analytical methods. High resolution mass spectrometry was recorded on an Agilent 6210 Time-of-Flight LC/MS system. Confirmation of molecular formula was accomplished using electrospray ionization in the positive mode with the Agilent Masshunter software (version B.02).

ML266: The reagents and solvents used were commercial anhydrous grade. They were used without further purification. The column chromatography was carried out over silica gel (100–200 mesh). 1H- and 13C NMR spectra were recorded on a Bruker 400 MHz spectrometer from solutions in CDCl3 and DMSO-d6. Automated preparative reverse-phase HPLC purification was performed using an Agilent 1200 Mass-Directed Fractionation system (prep pump G1361 with gradient extension, make-up pump G1311A, pH modification pump G1311A, HTS PAL autosampler, UV-DAD detection G1315D, Fraction Collector G1364B, and Agilent 6120 quadrapole spectrometer G6120A). The preparative chromatography conditions included a Waters X-Bridge C18 column (19 × 150mm, 5μm, w/19 × 10mm guard column), elution with a water and CH3CN gradient, which increases 20% in CH3CN content over 4 minutes at a flow rate of 20 mL/min (modified to pH 9.8 through addition of NH4OH by auxiliary pump), and sample dilution in DMSO. The preparative gradient, triggering thresholds, and UV wavelength were selected based on the analytical-scale HPLC analysis of each crude sample. The analytical method employed a Waters Acquity system with UV detection and mass detection (Waters LCT Premier). The analytical method conditions included a Waters Aquity BEH C18 column (2.1 × 50mm, 1.7μm) and elution with a linear gradient of 5% CH3CN in pH 9.8 buffered aqueous NH4OH to 100% CH3CN at 0.6 mL/min flow rate. The purity was determined using UV peak area at 214 nm.

2.1. Assays

Table 1Summary of Assays

PubChem AIDTypeTargetConc. RangeSamples TestedNotes
AID 2101Primary qHTSN370S GC57.5 μM – 0.7 nM326,770Tissue, blue
AID 2590ConfirmatoryN370S GC54 μM – 0.01 nM320Tissue, blue
AID 2613SecondaryN370S GC57.5 μM – 0.3 nM83Tissue, red
AID 2592SecondaryWildtype GC57.5 μM – 0.3 nM21Tissue, blue
AID 2588SecondaryWildtype GC50 μM – 0.1 nM152Tissue, red
AID 2595SecondaryWildtype GC77 μM – 0.3 nM52Purified, blue
AID 2597SecondaryN370S GC77 μM – 0.3 nM52Purified, blue
AID 2596SecondaryN370S GC230 μM – 0.1 nM94Purified, natural substrate
AID 2577Anti-targetAlpha-glucosidase57.5 μM – 0.3 nM70Purified, blue
AID 2578Anti-targetAlpha-galactosidase57.5 μM – 0.3 nM70Purified, blue
AID 2587TertiaryN370S GC100 μM – 10 nM4Immunostaining of fibroblast lysosomes
AID 2589TertiaryWildtype GC100 μM – 10 nM4Immunostaining of fibroblast lysosomes
AID 2593SummaryN370S GC

Fluorescent assays for gluocerebrosidase specific activity

The primary screening assay and many of the secondary assays are based on a fluorescent read-out and are direct enzymatic assays that measure GCase specific activity. Three different substrates are used: 4-methylumbelliferyl-β-D-glucoside (Ex365/Em440 “blue”), resorufin-β-D-glucoside (Ex573/Em590 “red”) and BODIPY tagged glucosylceramide (Ex505/Em540 “natural”) (Figure 1). Furthermore, four different sources of GCase are used for these assays; the N370S mutant (the most common mutation) and wildtype GCase, both from either tissue homogenate or purified recombinant enzyme. All of these assays and their data can be found in PubChem, linked to the summary AID 2593. Table 2 shows a general protocol that was used for these assays, and the details can also be found in PubChem.

Figure 1. Enzymatic reactions catalyzed by GCase in assays.

Figure 1

Enzymatic reactions catalyzed by GCase in assays.

Table 2. Typical protocol of primary and secondary assays.

Table 2

Typical protocol of primary and secondary assays.

A potential reason for the discrepancy in activity observed in assays that used the purified enzyme versus the spleen homogenate could be the presence of additional activating factors in the tissue. For instance, GCase activity is modulated in cells through the binding of the allosteric activator Saposin C.[1,2] In the GCase enzyme assay with purified enzyme, the addition of sodium taurocholate, a bile salt, is required to activate the enzyme.[3] It can be speculated that the variation in inhibitory activity between isolated enzyme and tissue homogenate is due to the differences between the active GCase conformation induced by detergent and the one induced by Saposin C and/or other factors in cells. These conformational differences might also explain why a chemical series with the capacity to activate and not inhibit the enzyme has not been previously found via conventional purified-enzyme screening assays. It may be that a detergent like sodium taurocholate induces a conformation that activates enzyme turnover to a maximum, and thus, further activation by a small molecule simply cannot be observed. In contrast, tissue homogenate conditions do not require the use of additional activating components, because natural activators like Saposin C are already present in the homogenate; thus, these conditions might provide a more faithful representation of the actual cellular activity.

Anti-target assays against alpha-glucosidase and alpha-galactosidase

To characterize compound selectivity, selected hits from the primary screen were screened against purified alpha-glucosidase and alpha-galactosidase, related sugar hydrolases. Alpha-glucosidase is responsible for hydrolysis of terminal, non-reducing 1,4-linked alpha-D-glucose residues with release of alpha-D-glucose, and alpha-galactosidase is a homodimeric glycoprotein that hydrolyzes the terminal alpha-galactosyl moieties from glycolipids and glycoproteins. This is a fluorogenic enzyme assay with 4-methylumbelliferyl-alpha-D-pyranoside and 4-methylumbelliferyl-alpha-D-galactopyranoside as the substrates, respectively. Upon the hydrolysis of this fluorogenic substrate, the resulting product, 1, 4-methyllumbelliferone, can be excited at 365 nm and emits at 440 nm. This excitation can be detected by a standard fluorescence plate reader. Data were normalized to the controls for basal activity (without enzyme) and 100% activity (with enzyme). The AC50 values were determined from concentration-response data modeled with the standard Hill equation. Assay buffer: 50 mM citric acid (titrated with potassium phosphate to pH 5.0), 0.005% Tween-20, pH 5.0. pH 5.0 is an optimal condition for this enzyme assay. Table 2 shows a general protocol of these experiments, and the details can be found in PubChem.

Chaperone translocation experiments in human fibroblasts

This assay attempts to quantitate translocated glucocerebrosidase protein in patient-derived fibroblasts following extended compound incubation. The fibroblasts tested in this experiment were homozygous either for N370S glucocerebrosidase or wildtype GCase. Primary dermal fibroblasts derived from skin biopsies from two previously described N370S/N370S Gaucher patients and a control were seeded in Lab-Tek 4 chamber slides (Fisher Scientific, Pittsburgh, PA). After compound treatment, fibroblasts were fixed in 3% paraformaldehyde. The cells were permeabilized with 0.1 % Triton-X for 10 min. and blocked in PBS containing 0.1% saponin, 100 μM glycine, 0.1% BSA and 2% donkey serum. This was followed by an incubation with mouse monoclonal anti-LAMP1 or LAMP-2 (1:100, Developmental Studies Hybridoma bank, University of Iowa, Iowa City, IA) and the rabbit polyclonal anti-GCase R386 antibody (1:500); the cells were washed and incubated with secondary donkey anti-mouse or anti-rabbit antibodies conjugated to ALEXA-488 or ALEXA-555, respectively (Invitrogen, Carlsbad, CA), washed again, and mounted in VectaShield with DAPI (Vector Laboratories, Burlingame, CA) (Table 3).

Table 3. Protocol of fibroblast translocation experiment.

Table 3

Protocol of fibroblast translocation experiment.

Cells were imaged with a Zeiss 510 META confocal laser-scanning microscope (Carl Zeiss, Microimaging Inc., Germany) using an Argon (458, 477, 488, 514 nm) 30 mW laser, a HeNe (543 nm) 1 mW laser, and a laser diode (405 nm). Low and high magnification images were acquired using a Plan-Apochromat 20X/0.75 objective and a Plan-Apochromat 100x/1.4 oil DIC objective, respectively. Images were taken with the same laser settings and all the images shown are collapsed z-stacks.

LC-MS hydrolysis experiment

This assay assures that any compound autofluorescence does not interfere with activity in the fluorescence-based primary and secondary assays. A liquid chromatography assay is linked to a mass spectrometer to assess the ability of glucocerebrosidase from spleen homogenate to cleave either 4-methylumbelliferyl-beta-D-glucopyranoside substrate or a labeled version of the natural substrate, glucosylceramide. Both substrates have a pro-fluorescent tag, which allows the product fraction to be easily identified with liquid chromatography; however, identification of the reaction product is done using the mass spectrometer. This tissue homogenate assay most closely reflects physiological conditions in the body.

Chromatography was performed using an Agilent HPLC. The Agilent 1200 LC was equipped with a quaternary pump, a G1315 diode array detector, and a G1321 Fluorescent Detector. A 4.6 × 250 mm Agilent Eclipse Plus C18 (5 micron) at ambient temperature was used at a flow rate of 1.8 mL/Min with a gradient of 85/15 (methanol/0.1% formic acid in water) to 100 methanol over 10 minutes. The BODIPY tagged natural substrate was monitored using fluorescence detection with an excitation wavelength of 505 nanometers and emission wavelength at 540 nanometers.

2.2. Probe Chemical Characterization

ML198

Probe Characterization of ML198.

Probe Characterization of ML198

*Purity >95% as determined by LC/MS and 1H NMR analyses.

N-(4-Ethynylphenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidine-3-carboxamide

LC-MS Retention Time = 2.61 min; 1H NMR (400 MHz, DMSO-d6) δ ppm 2.71 (s, 3 H), 2.77 (s, 3 H), 4.11 (s, 1 H), 7.21 (s, 1 H), 7.49 (d, J=8.6 Hz, 2 H), 7.77 (d, J=8.6 Hz, 2 H), 8.65 (s, 1 H), 10.31 (s, 1 H); HRMS (ESI) m/z (M+H)+ calcd. for C17H15N4O, 291.1240; found 291.1245.

Figure 2. RP HPLC/UV/MS chromatogram for the probe compound ML198.

Figure 2RP HPLC/UV/MS chromatogram for the probe compound ML198

Figure 3. List and structures of probe and analogs that have been submitted to the MLSMR.

Figure 3List and structures of probe and analogs that have been submitted to the MLSMR

In limited stability studies in PBS buffer over 47 hrs (Figure 4), we did not identify any decomposition product.

Figure 4. Stability of ML198 in D-PBS pH 7.4 at room temperature over 48 hr.

Figure 4

Stability of ML198 in D-PBS pH 7.4 at room temperature over 48 hr.

Scheme 1. Synthetic route to ML198.

Scheme 1Synthetic route to ML198

ML266

Probe Characterization of ML266.

Probe Characterization of ML266

*Purity >95 % as determined by LC/MS and 1H NMR analysis.

2-(2-((4-bromophenyl)amino)-2-oxoethoxy)-N-(2-(methyl(phenyl)amino)-2-oxoethyl)benzamide: LC-MS Retention Time: 3.295 min; 1H NMR (400 MHz, DMSO) δ 10.44 (s, 1H), 8.84 (t, J = 4 Hz, 1H), 7.73 (d, J = 5.6 Hz, 1H), 7.68 (d, J = 6.8 Hz, 2H), 7.50 (d, J = 5.6 Hz, 5H), 7.44 (bs, 3H), 7.17 (d, J = 6.8 Hz, 1H), 7.07 (t, J = 6.4 Hz, 1H), 4.89 (s, 2H), 3.19 (s, 3H) ppm; 13C NMR (100 MHz, DMSO) δ 167.7, 166.6, 165.5, 155.7, 137.7, 132.5, 131.6, 130.3, 129.8, 128.0, 127.3, 123.2, 121.5, 115.4, 113.8, 67.7, 41.8, 36.9 ppm; IR νmax (cm−1) 3273, 3061, 2924, 1637, 1594; HRMS calcd for C24H22BrN3O4 [M+H+] 496.0866, found 496.0876.

Figure 5. RP HPLC/UV/MS chromatogram for the probe compound ML266.

Figure 5RP HPLC/UV/MS chromatogram for the probe compound ML266

Figure 6. List and structures of probe and analogs that have been submitted to the MLSMR.

Figure 6List and structures of probe and analogs that have been submitted to the MLSMR

ML266 demonstrates good stability in PBS buffer for 48 hrs (Figure 7).

Figure 7. Stability of ML266 in D-PBS pH 7.4 buffer at room temperature over 48 hr.

Figure 7

Stability of ML266 in D-PBS pH 7.4 buffer at room temperature over 48 hr.

Scheme 2. Synthetic route to ML266.

Scheme 2Synthetic route to ML266

2.3. Probe Preparation

ML198

Preparation of N-(4-Ethynylphenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidine-3-carboxamide (ML198)
Image ml266fu4

Step 1: Pentane-2,4-dione (1.46 mL, 14.2 mmol) and ethyl 3-amino-1H-pyrazole-4-carboxylate (2.00 g, 12.9 mmol) were heated in a sealed tube with acetic acid (10 mL) at 110 °C overnight. The reaction reached completion by LCMS (LC-MS: rt (min) = 3.08). The acetic acid was removed by blowing air, with the flask heated to 75 °C. The crude residue of ethyl 5,7-dimethylpyrazolo[1,5-a]pyrimidine-3-carboxylate (assumed to be 12.89 mmol) was suspended in MeOH (15 mL) and treated with 7.2 M sodium hydroxide (5.37 mL, 38.7 mmol). The mixture was heated to 80 °C (at this temperature the solid dissolved) and then stirred for 3 hrs. The reaction was cooled and the neutralized to pH 6–7. The slurry was filtered via a Büchner funnel under house vacuum, and the solid residue was washed with water and then diethyl ether to obtain 5,7-dimethylpyrazolo [1,5-a]pyrimidine-3-carboxylic acid (1.4 g, 7.3 mmol, 57 % yield). LC-MS: rt (min) = 2.61. 1H NMR (400 MHz, DMSO-d6) δ ppm 2.57 (s, 3 H), 2.71 (s, 3 H), 7.10 (s, 1 H), 8.50 (s, 1 H).

Image ml266fu5

Step 2: 5,7-Dimethylpyrazolo[1,5-a]pyrimidine-3-carboxylic acid (722 mg, 3.78 mmol), 4-ethynylaniline (442 mg, 3.78 mmol), and HATU (1436 mg, 3.78 mmol) were taken up in DMF (10 mL) and then treated with diisopropylethylamine (1.979 mL, 11.33 mmol). The contents stirred at rt overnight. The product had precipitated from reaction mixture. The reaction was diluted with water, filtered through a Büchner funnel under house vacuum. The residue was washed with water (X2), then CH2Cl2, then diethyl ether, and air dried to obtain N-(4-ethynylphenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidine-3-carboxamide (500 mg, 1.72 mmol, 46 % yield). LC-MS: rt (min) = 3.65. 1H NMR (400 MHz, DMSO-d6) δ ppm 2.71 (s, 3 H), 2.77 (s, 3 H), 4.11 (s, 1 H), 7.21 (s, 1 H), 7.49 (d, J=8.6 Hz, 2 H), 7.77 (d, J=8.6 Hz, 2 H), 8.65 (s, 1 H), 10.31 (s, 1 H).

ML266

Preparation of 2-(2-((4-bromophenyl)amino)-2-oxoethoxy)-N-(2-(methyl(phenyl)amino)-2-oxoethyl)benzamide (ML266)

Step 1: 4-Bromoaniline (4.12 g, 23.95 mmol) was dissolved in 50 mL of dichloromethane in a round bottomed flask containing a stir bar. While stirring, triethylamine (2.91 g, 28.74 mmol) was added to the reaction mixture and then cooled to 0 °C. 2-Chloroacetyl chloride (2.71 g, 23.95 mmol) was added dropwise to the reaction mixture, which was then allowed to slowly warm to room temperature and stir for 16 hours. The reaction mixture was diluted with 100 mL of dichloromethane, washed twice with 1N HCl, run through a silica gel plug, and then concentrated in vacuo to give N-(4-bromophenyl)-2-chloroacetamide (5.74 g, 97 % yield).

Step 2: Potassium carbonate (3.75 g, 27.1 mmol) was added to a mixture of N-(4-bromophenyl)-2-chloroacetamide (2.25 g, 9.05 mmol) and methyl 2-hydroxybenzoate (1.38 g, 9.05 mmol) in DMF (30 mL). The reaction mixture was stirred for 16 hours. 1 N HCl was added slowly to the reaction mixture. The combined solution was extracted three times with ethyl acetate. The resulting organic layer was washed twice with water, once with brine, run through a silica plug, and then concentrated in vacuo to give methyl 2-(2-((4-bromophenyl)amino)-2-oxoethoxy)benzoate (3.11 g, 94 % yield).

Step 3: Methyl 2-(2-((4-bromophenyl)amino)-2-oxoethoxy)benzoate was dissolved in a mixture of tetrahydrofuran and methanol (2:1). Potassium hydroxide (.82 g, 14.55 mmol) was dissolved in 5 mL of water and then added to the reaction mixture. This mixture was then allowed to stir for 16 hours. Water was added. The aqueous solution was then extracted with diethyl ether, and the organic phase was discarded. The aqueous phase was then placed in a round bottomed flask with a stir bar, cooled in an ice bath, and then the pH was adjusted to approximately 3. A precipitate resulted that was immediately filtered off and dried to give 2-(2-((4-bromophenyl)amino)-2-oxoethoxy)benzoic acid (.96 g, 94 % yield).

Step 4: 2-(2-((4-Bromophenyl)amino)-2-oxoethoxy)benzoic acid (0.19 g, 0.55 mmol) was dissolved in 10 mL of dichloromethane in a round bottomed flask containing a stir bar. N,N-dimethylformamide (0.04 g, 0.549 mmol) was added to this stirring mixture, and then the mixture was cooled to 0 °C. A solution of oxalyl chloride (0.209 g, 1.65 mmol) in 3 mL of dichloromethane was added dropwise. The reaction was allowed to slowly warm to room temperature and then to further mix for one hour. The reaction was concentrated in vacuo to remove solvent and excess oxalyl chloride, while not heating over 30 °C. This crude mixture was then redissolved in 2 mL of dichloromethane and added dropwise to a solution of 2-amino-N-methyl-N-phenylacetamide (0.12 g, 0.60 mmol), triethylamine (0.12 g, 1.21 mmol) in dichloromethane (2 mL) that had been previously placed in Metler-Toledo Bohdan Miniblock™ reaction tube (Metler-Toledo Autochem Reaction tubes 10.0 mi Part # 1352118) (Note: 6 × 4 Miniblock™ setups were used to generate 24 different products per block in parallel). After the addition, the septum layer and cover plate were secured onto the Miniblock™ with spring clamps. The block was then secured onto a Bohdan Miniblock™ Compact Shaking and Washing Station, in which the shaker was set at 600 rpm for 16 hours. The Miniblock™ was then removed from the shaker, followed by a subsequent draining of the reaction mixture into a second Miniblock™ containing a Biotage ISOLUTE ® SPE Accessories Phase Separator Tube (Part # 120–1905-CG), containing a 1N HCl solution (3 mL). A cover plate was placed on the second Miniblock™ containing the reaction mixture, and then the Miniblock™ was placed on the shaker and was allowed to shake for five minutes at 600 rpm. After removal of the Miniblock™ from the shaker, the organic phase was allowed to drain into a sample collection tube. Sample was concentrated in vacuo in a GeneVac HT-4X centrifugal evaporator and then purified via automated preparative reverse-phase HPLC purification to give 2-(2-((4-bromophenyl)amino)-2-oxoethoxy)-N-(2-(methyl(phenyl)amino)-2-oxoethyl) benzamide (0.15 g, 43 % yield).

3. Results

3.1. Dose Response Curves for Probe

The activity profile of the probe molecules are shown in Figure 8 along with isofagomine, the small molecule inhibitor chaperone for GCase.

Figure 8. Concentration response profiles for probes ML198 and ML266 using 4-methylumbelliferyl-β-D-glucopyranoside (blue) as the substrate, with N370S spleen homogenate.

Figure 8

Concentration response profiles for probes ML198 and ML266 using 4-methylumbelliferyl-β-D-glucopyranoside (blue) as the substrate, with N370S spleen homogenate.

3.2. Cellular Activity

Figure 9. Human Fibroblast experiment: the nucleus is stained with DAPI (blue), the lysosome is stained with LAMP-2 (green), and GCase is visualized through a GBA antibody (red).

Figure 9Human Fibroblast experiment: the nucleus is stained with DAPI (blue), the lysosome is stained with LAMP-2 (green), and GCase is visualized through a GBA antibody (red)

With DMSO treatment, GCase remains largely in the nucleus, and only 5% of the cells indicated translocation of GCase to the lysosome. We tested the known iminosugar isofagomine and observed translocation in 17% of the cells at 5 μM. At 5 μM, ML198 and ML266 give very convincing translocation visually, and their percentage of cell translocations are 20 and 15%, respectively.

The high magnification images in Figure 10 (the yellow color in the bottom right quadrant) clearly show the co-localization of CGase in the periphery of the cell, where the lysosome is located. The colocalization was also observed to increase in a dose-dependent manner, and for other active analogs in the series (not shown), thereby demonstrating the chaperone behavior of the series.

Figure 10. High magnification image of translocation experiment in a single cell with with 5 μM ML198.

Figure 10

High magnification image of translocation experiment in a single cell with with 5 μM ML198. Localization of GCase (red) into the lysososomes (green) is evident by the yellow color in the overlapped image in the bottom right quadrant.

The selection of the probe candidate ML266 was facilitated by the lack of toxicity at 20 μM. This molecule was also evaluated at 5 μM, where 14% of the N370S Gaucher patient fibroblasts exhibited increased co-localization of GCase at the lysosome. This is evident in the high magnification image in Figure 11, where bright yellow staining is observed at the periphery of the cells where mature lysosomes are located. Most of the other compounds in the series exhibited cytotoxicity at 10 and 20 μM.

Figure 11. High magnification image of translocation experiment in a single cell with 5 μM ML266.

Figure 11

High magnification image of translocation experiment in a single cell with 5 μM ML266. Localization of GCase (red) into the lysososomes (green) is evident by the yellow color in the overlapped image in the bottom right quadrant.

3.3. Profiling Assays

The probe compounds were profiled against β-glucosidase and β-galactosidase. They were found to be inactive against these glucosidases.

The microsomal stability and Caco-2 permeability data for ML198 are promising (Table 4). The probe molecule has low water solubility, however. The probe ML266 has also been evaluated in ADME type assays, also (Table 4). It shows modest permeability in a Caco-2 assay and has good water solubility. However, analysis of metabolic stability after a 60 min-incubation in mouse liver microsomes revealed rapid clearance. This issue is being addressed.

Table 4. ADME profile for ML198 and ML266.

Table 4

ADME profile for ML198 and ML266.

4. Discussion

4.1. Comparison to Existing Art and How the New Probe is an Improvement

Approved small molecule therapy towards Gaucher disease is restricted to substrate reduction therapy with Zavesca® (miglustat, Figure 12). Other iminosugars (e.g. NN-DNJ and isofagomine, Figure 12) that have been described in the literature are inhibitors which also act as chaperones. The class of pyrazolpyrimidines and salicylic acid derivatives described in this report demonstrates biochemical activation of CGase, as well as chaperone activity in translocation assays. These molecules are first-in-class small molecule activators of GCase, unlike current prior art which consists of iminosugars (Figure 12) or other classes of inhibitors (Figure 13). They have the unique advantage over existing chaperones in that they are not inhibitors of the enzyme. Thus, the extra burden towards resumption of catalytic activity after GCase translocation to the lysosome is not an issue. Though the AC50s for these compounds may be at micromolar concentrations, it is not clear what in vivo exposure will be needed and for what duration to have a therapeutic effect. As we know via the enzyme replacement therapy, even a slight correction of the enzyme function can lead to very positive therapeutic effects. Therefore, these compounds hold tremendous potential towards defining a new direction in the further development of chaperone therapy for Gaucher disease (Table 5).

Figure 12. Clinically relevant small molecules for Gaucher disease.

Figure 12

Clinically relevant small molecules for Gaucher disease.

Figure 13. Representative non imino sugar chaperone inhibitors discovered at the NIH Chemical Genomics Center.

Figure 13

Representative non imino sugar chaperone inhibitors discovered at the NIH Chemical Genomics Center.

Table 5. Comparison of probe to prior art.

Table 5

Comparison of probe to prior art.

Table 6Inhibitory profile of GCase chaperone inhibitors illustrated in Figure 13

CIDPurified GCaseSpleen Homogenate
Wt
IC50 μM
N370S
IC50 μM
wt
IC50 μM
N370S
IC50 μM
CID-6503780.40117.9Inactive
CID-50672810.030.562.23.2–12.6*
CID-22102900.101.63.2Inactive
Ml1558.9Inactive0.280.33
ML1560.712.50.360.58

The data here represents inhibition of turnover of methyl-umbelliferyl β-D-glucopyranoside (4MU-β-Glu,) as the substrate and highlights the critical nature of the assay format for the evaluation of a particular structural class of compounds.

5. References

1.
John M, Wendeler M, Heller M, Sandhoff K, Kessler H. Characterization of Human Saposins by NMR Spectroscopy. Biochemistry. 2006;45:5206–5216. [PubMed: 16618109]
2.
Vaccaro AM, Salvioli R, Barca A, Tatti M, Ciaffoni F, Maras B, Siciliano R, Zappacosta F, Amoresano A, Pucci P. Structural analysis of saposin C and B. Complete localization of disulfide bridges. J Biol Chem. 1995;270:9953–9960. [PubMed: 7730378]
3.
Motabar O, Goldin E, Leister W, Liu K, Huang W, Marugan JJ, Sidransky E, Zheng W. A high throughput glucocerebrosidase assay using the natural substrate glucosylceramide. Anal Bioanal Chem. 2011. in press. [PMC free article: PMC3351006] [PubMed: 22033823]

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