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.

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.

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.

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

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.

Scheme 1Synthetic route to ML198

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 ν_max (cm⁻¹) 3273, 3061, 2924, 1637, 1594; HRMS calcd for C₂₄H₂₂BrN₃O₄ [M+H⁺] 496.0866, found 496.0876.

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

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.

Scheme 2Synthetic route to ML266

ML198

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

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).

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