In this report, ML155 (CID 40225210) is described as a probe that is able to inhibit the hydrolytic activity of the N370S mutant form of glucocerebrosidase, as well as wild type glucocerebrosidase, in tissue homogenate assays. The probe does not inhibit purified glucocerebrosidase, but the cellular activity of the enzyme is known to be dependent on interactions with other factors, such as Saposin C. Importantly, the probe increased glucocerebrosidase translocation to the lysosome in Gaucher patient-derived fibroblasts homozygous for the N370S mutation, and can be used to study ER-lysosomal trafficking of clinically relevant GC mutants in vitro. This probe may be a useful lead for the clinical development of a chemical chaperone of glucocerebrosidase.

Probe Structure & Characteristics

ML155

Recommendations for scientific use of the probe

The probe is able to inhibit the hydrolytic activity of the N370S mutant form of glucocerebrosidase, as well as wild type glucocerebrosidase, in tissue homogenate assays. The probe does not inhibit purified glucocerebrosidase, but the cellular activity of the enzyme is known to be dependent on interactions with other factors, such as Saposin C. Importantly, the probe increased glucocerebrosidase translocation to the lysosome in Gaucher patient-derived fibroblasts homozygous for the N370S mutation, and can be used to study ER-lysosomal trafficking of clinically relevant GC mutants in vitro. This probe may be a useful lead for the clinical development of a chemical chaperone of glucocerebrosidase.

1. Introduction

Gaucher disease is an autosomal recessive disorder resulting from mutations in the enzyme Glucocerebrosidase (EC 3.2.1.45; also known as acid beta-glucosidase) and affects 1 in 50,000 live births (1). The function of Glucocerebrosidase (GC) is to hydrolyze the beta glucosidic linkage of glucocerebrosidases, also called glucosylceramides (2). These glycosphingolipids are cell membrane components that maintain the stability of the lipid bilayer, function as cellular recognition elements and play an important role in cellular adherence (3). The deficiency of GC due to the genetic mutations results in the accumulation of glucosylceramides in lysosomes.

There are more than 200 recognized mutations of the GC gene (4). Although many GC mutants are still functional (5), conformational differences in mutant proteins reduce their recognition by transporters, resulting in ER protein accumulation and premature degradation in endosome. The inability of mutant proteins to reach their site of action causes lysosomal accumulation of glucosylceramides and lysosomal enlargement. Ultimately, this leads to liver and spleen enlargements, as well as the neurological symptoms in type 2 and type 3 Gaucher disease.

Currently, the only FDA approved treatment for Gaucher disease is enzyme replacement therapy using the human recombinant glucocerebrosidase, Cerezyme. Although this approach does address some aspects of the disease, the limited tissue distribution of the infused enzyme, for example poor CNS penetration, reduces its therapeutic benefits in type 2 and type 3 Gaucher patients (6). Small molecule chaperone therapy has been proposed as an alternative therapeutic strategy for Gaucher disease. The binding of small molecules to mutant protein may facilitate proper folding and translocation of the mutant protein to the lysosome (7–8). Several imino sugar inhibitors of glycosidases have been reported to have chaperone activity (9–20). Isofagomine is an imino sugar that can act as a chaperone for glucocerebrosidase, but was recently withdrawn from clinical trial testing.

Imino sugars inhibit glycosidases by mimicking the transition state of the glycosidic cleavage, and as such tend to be poorly selective (13). Therefore, it would be desirable to develop alternative, more selective series with chaperone activity. It is also important to remark that molecules with very potent inhibitors may have a lower probability of ultimately producing a therapeutic effect or an increase in GC activity in the lysosome. Though the GC protein translocation is increased, the tightly bound inhibitors may not be easily displaced by the natural substrate, and may even inhibit any residual activity of the enzyme (2), (8), (21). Previous chaperone molecules that have entered clinical trials are all potent GC inhibitors. Our goal is to identify a non-imino sugar series with modest inhibitory capacity and good chaperone activity.

GC activity is modulated in cells through the binding of an allosteric activator, Saposin C (27). In the GC enzyme assay with a purified enzyme, the addition of sodium taurocholate, a bile salt, is required to activate the enzyme (23). Assays by our group and others that screen for inhibitors of GC using purified enzyme have identified several interesting inhibitor chemotypes, but most have reduced or no activity when tested using tissue homogenate that contains both GC and Saposin C, as well as other components that may be required for GC function in cells. We speculate that this variation in activity is due to the GC conformational differences between the active conformation induced by detergent and the one induced by Saposin C and/or other factors in cells. Additionally, 70% of Gaucher patients carry the N370S mutation, and therefore we developed an assay utilizing the spleen (28) homogenate derived from a GC patient homozygous for the N370S mutation in the primary screen. This assay uses a fluorogenic substrate, 4-methylumbelliferone β-D-glucopyranoside, to monitor glucoerebrosidase specific activity, and the spleen tissue homogenate as the GC enzyme preparation. Upon hydrolysis, 4-methylumbelliferone (4-MU) is liberated, which produces a fluorescent emission at 440 nm when excited at 370 nm.

2. Materials and Methods

Unless otherwise stated, all reactions were carried out under an atmosphere of dry argon or nitrogen in dried glassware. Indicated reaction temperatures refer to those of the reaction bath, while room temperature (rt) is noted as 25^oC. All solvents were of anhydrous quality purchased from Aldrich Chemical Co. and used as received. Commercially available starting materials and reagents were purchased from Aldrich, TCI and Acros and were used as received. Analytical thin layer chromatography (TLC) was performed with Sigma Aldrich TLC plates (5x 20cm, 60 Å, 250μm). Visualization was accomplished by irradiation under a 254nm UV lamp.

Chromatography on silica gel was performed using forced flow (liquid) of the indicated solvent system on Biotage KPSil pre-packed cartridges and using the Biotage SP-1 automated chromatography system. ¹H NMR spectra were recorded on a Varian Inova 400 MHz spectrometer. Chemical shifts are reported in ppm with the solvent resonance as the internal standard (CDCl₃ 7.27 ppm, DMSO-d₆ 2.49 ppm, for ¹H NMR). Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, sep = septet, quin = quintet, br = broad, m = multiplet), coupling constants, and number of protons. Low resolution mass spectra (electrospray ionization) were acquired on an Agilent Technologies 6130 quadrupole spectrometer coupled to an Agilent Technologies 1200 series HPLC. The HPLC retention times were recorded through short standard gradient 4% to 100% acetonitrile (0.05% TFA) over 3 minutes (t₁) or long standard gradient 4% to 100% acetonitrile (0.05% TFA) over 7 minutes (t₂) using Luna C₁₈ 3 micron 3x 75mm column with a flow rate of 0.800 ml/min. High resolution mass spectral data was collected in-house using an Agilent 6210 time-of-flight mass spectrometer, also coupled to an Agilent Technologies 1200 series HPLC system.

2.3. Probe Preparation

With the confirmatory data in hand, we embarked on systematic SAR modifications based on commercially available quinazolines as shown in Table 1. Scheme 1 discloses the synthesis strategy for some of the modifications at the quinazoline core (26).

Table 1

SAR of commercial quinazolines.

Scheme 1

General strategy for modification in the functional core.

Commercially available Boc protected piperazine 14 was sulphonytaled, using the sulphonyl chloride 15 in the presence of a suitable base such as Diisopropylethylamine, followed by a quantitative deprotection of the Boc functional group to yield intermediate 16. The next step involves the selective chloro displacement at the core ring 17 to produce compound 18, followed by a Suzuki cross-coupling reaction with an aromatic or heteroaromatic boronic acid 19 to produce final compound 20. Application of this synthetic strategy allowed us the synthesis of analogs having quinazoline, pyrimidine and isoquinoline cores.

The synthesis of analogs with a purine core, Scheme 2, required the selective protection of the ring NH with a methyl pivalate functional group, followed by cross coupling and deprotection. Numerous attempts of carrying out Suzuki reactions with the free purine core or using a Boc protecting group failed to yield the intended coupling product.

Scheme 2

Synthesis of purine analogs.

For the SAR study of the sulphonamide portion of the molecule, we modified the synthetic procedure toward a more convenient methodology that allowed us to introduce variation at the last step of the synthesis. Scheme 3 shows how sulphonylation, carboxylic coupling and alkylation of the key intermediate 32 provided a variety of final compounds.

Scheme 3

Synthesis of analogs with modification of the aromatic sulphonamide.

We also synthesized a number of analogs testing the flexibility of the diamine linker, as well as the activity of other amine-linked functionalities attached to the piperidine ring. Schemes 4 and 5 present example syntheses of some of these compounds, which were performed in a similar fashion to the previously described compound syntheses.

Scheme 4

Synthesis of analogs with a flexible linker.

Scheme 5

Synthesis of additional analogues with a flexible linker.

Regarding the synthesis of analogs with aromatic and heteroaromatic modifications at the 2 position, Scheme 6 shows the synthetic methodology involving the production of the organotin 63 as a convenient intermediate for last step diversification using Stille coupling. Thus, 2,4 quinazoline dione, 60, was converted in 2,4-dibromoquinazoline 61 with phosphorus oxybromide. Selective halogen displacement with substituted piperidine 21 and Lithium halogen exchange followed by reaction with tributyl tin chloride yielded compound 63. Cross coupling reaction between 63 and heteroaromatic halogens yielded final compounds 64.

Scheme 6

Synthesis of analogs with modifications at the 2 position.

Additional modifications at the same position 2 were obtained as show in Schemes 7 and 8. Thus, cross-coupling reaction with Zinc cyanide catalyzed by Palladium yielded intermediate 66. Hydrolysis and esterification of nitrile 66 yielded final compounds 67–69. Reduction of starting material 65 produced compound 70, and displacement of the chloro at position 2 yielded final compound 71.

Scheme 7

Additional modifications at the 2 position.

Scheme 8

Synthesis of thiophene analogs with improved solubility.

3. Results

3.2. Dose Response Curves for Probe

Figure 1Inhibitory curves of compound 13 (CID 40225210), against spleen homogenate homozygous for N370S GC using 4-methylumbelliferyl-β-D-glucopyranoside (curve in black) and resorufin β-D-glucopyranoside as substrates (curve in blue), and wildtype spleen homogenate using 4-methylumbelliferyl-β-D-glucopyranoside (curve in cyan; AID 2592)

3.4. SAR Tables

Tables 3–8 show the capacity of compounds to inhibit the hydrolysis of 4-methylumbelliferone β-D-glucopyranoside. As a positive control, we measured the activity of isofagomine in the same assay (AC50 = 0.080μM).

Table 3

Analogs with sulfonamide aromatic modifications having a phenyl at the two position.

Table 4

Analogs with sulfonamide aromatic modifications having a thiophene at the two position.

Table 5

A continuation of Table 4 - analogs with sulfonamide aromatic modifications having a thiophene at the two position.

Table 6

Analogs with the modifications at the linker.

Table 7

Analogs with modification at the 2 position of the quinazoline core.

Table 8

Analogs with modifications at the molecular core.

3.5. Cellular Activity

Compound 13 was characterized in a direct assay of chaperone activity using patient fibroblasts; that is, we measured 13’ s ability to increase the translocation of GC to the lysosome (8), (16), (24), (25). Briefly, fibroblasts obtained from a Gaucher patient homozygous for the N370S mutation and from another donor with wildtype GC were incubated for five days with NCGC00182292 (CID 40225210), in a range of concentrations from 1nM to 50μM, followed by cell fixation and staining with a selective fluorescent GC-antibody (AID2587 and AID2589). Compounds able to increase the ER-Lysosomal traffic show increased fluorescent GC-antibody staining in lysosomes. DMSO and Isofagomine were use as negative and positive controls. Figure 2 shows the increment of signal in produced by 13, thereby confirming the chaperone activity of this compound.

Figure 2

Chaperone activity of compound 13, NCGC00182292 (CID 40225210), and others using wildtype and homozygous, mutant N370S GC fibroblasts. Two genotypes of fibroblasts, fibroblasts homozygous for wildtype GC (top) and fibroblasts homozygous for N370S GC (bottom) (more...)

4. Discussion

The primary screen for this project assayed for GC enzyme in the context of spleen homogenate derived from a N370S Gaucher patient by monitoring the production of a fluorogenic substrate, 4-methylumbelliferone β-D-glucopyranoside. In addition, several other variations of GC enzyme assays were developed to help eliminate the false positive compounds in the primary screen, including a similar enzyme assay with a red-shifted fluorogenic substrate (resorufin-β-D-glucopyranoside) and an enzyme assay with a native substrate (glucosylceramide). Unfortunately, cleavage of glucosylceramide can only be done in the context of purified enzyme, as this enzyme assay uses a coupled enzyme reaction for detection in which the glucose oxidase/amplex red interacts with glucose, the product of glucosylceramide cleavage, to produce a fluorogenic signal. The strengths and weaknesses of these assays are described in detail below. In all the assays, activators and auto-fluorescent compounds may both give an apparent increase in signal and quenchers may give a decrease in signal. To eliminate nonspecific compounds, counterscreen enzyme assays with alpha-galactosidase and alpha-glucosidase were used. Activators and inhibitors were then confirmed using the primary screening assays as well as glucocerebrosidase assays using a red-shifted fluorogenic substrate, and product formation was also directly tracked using LC/MS. Once activity was confirmed, SAR studies were undertaken to characterize chemotypes in more detail.

Figure 3Principles of enzyme reactions and product spectrums of two GC enzyme assays

(a) The “Red” GC enzyme assay. The pro-fluorescent substrate Res-β-glucopyranoside is hydrolyzed to form two products, glucose and resorufin, with an excitation perk of 573 nm and an emission perk of 590 nm. This assay is used for the primary screen. (b) The “Blue” GC enzyme assay. The pro-fluorescent substrate 4MU-β-Glc is hydrolyzed to form two products, glucose and 4MU, with an excitation perk of 365 nm and an emission peak of 440 nm.

An inhibitory chemical probe for this project is defined as a molecule having EC₅₀ values in lower μM in the cell-based homogenate assay, and being selective against other glycosidic hydrolases (selective – EC₅₀ values > 10 fold against α-galactosidase and α-glucosidase).

From the primary screen, we identified several series of modulators, including several imino sugar molecules previously reported to inhibit the enzyme such as isofagomine (Table 9).

Table 9

Compound representatives of initial hit series. Isofagomine is an imino sugar previously reported to chaperone glucocerebrosidase. NCGC00092410 (CID 5067281) is a non-imino sugar previously reported to inhibit purified glucocerebrosidase (23).

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We evaluated several tissues finding that the spleen has one of the highest levels of GC activity.