The handle
http://hdl.handle.net/1887/87273
holds various files of this Leiden University
dissertation.
Author: Lahav, D.
49
3
FluoPol ABPP on Human Lysosomal β-Glucosylceramidase Identifies
Inhibitors from the LOPAC Library
3.1 Introduction
Glucosylceramide (GlcCer) is synthesized by glucosylceramide synthase (GCS) from UDP-glucose and ceramide. The biosynthesis of GlcCer takes place at the cytosolic leaflet of the Golgi apparatus. GlcCer is translocated into the lumen of the Golgi, where the carbohydrate structure is elongated into a more complex glycosphingolipid (GSL). Upon completion of the biosynthesis these complex structures are transported to the plasma membrane. After endocytosis of the plasma membrane and endosomal maturation to lysosomes the catabolic process, that is the stepwise degradation of complex GSLs, occurs.1 As the penultimate step, lysosomal retaining β-glucosylceramidase (GBA1) hydrolyzes glucosylceramide (GlcCer) to produce glucose and ceramide. GBA1 is a member of the glycoside hydrolase (GH) family, GH30 (www.cazy.org).2 The non-lysosomal retaining β-glucosylceramidase (GBA2), subject of studies reported in Chapter 2, is complementary to the activity of GBA1 and responsible for GlcCer breakdown outside the lysosomes.3,4 Glucosylceramide metabolism is summarized in Figure 1.
Figure 1: Simplified reaction scheme for the biosynthesis of GSLs and catabolic reactions performed
50
Mutations in the GBA1 gene generates the most common lysosomal storage disorder, Gaucher disease.5,6 Deficiency in enzyme activity results in the accumulation of GlcCer especially in macrophages. The presence of lipid-laden macrophages leads to a variety of symptoms, such as organomegaly and splenomegaly, but also to skeletal disfiguration and neuropathological manifestations.7 The severity and acuteness of these symptoms depend on the mutation within the GBA1 gene: some mutations lead to a mild phenotype, whereas others are much more severe or even lethal at birth.8 In addition, genetic mutations in GBA1 are a major risk factor for Parkinson’s disease (PD) and Lewy-body dementia.9 Recent studies show a link between impaired GBA activity and the development of α-synuclein accumulation, which attributes to the loss of nerve cells. While the underlying basis of this relationship remains elusive, it is reported that people who do not develop Gaucher disease, but are carriers of one defective gene copy, have a 7- to 10-fold risk of developing PD.10
Enzyme replacement therapy (ERT) is an effective treatment for the non-neuropathic Gaucher disease type 1. In ERT recombinant enzyme is intravenously administrated to patients. A small-molecule based therapy has reached the clinic as well and is termed substrate-reduction therapy (SRT). Whereas in ERT impaired endogenous enzyme is supplemented by recombinant enzyme with the aim to reach normal (as in healthy individuals) levels of glucosylceramide turnover, in SRT the synthesis of GlcCer by GCS is lowered to such a level that the impaired endogenous GBA1 levels can handle turnover of the remaining GlcCer.11,12 Neither of the two therapies is used for the treatment of Gaucher types 2 and 3, likely due to the neuropathological manifestations in these disease forms.13 Another small-molecule based treatment currently in clinical investigations is called pharmacological chaperone therapy (PCT). Pharmacological chaperones are small molecules that are intended to stabilize protein fold in endoplasmic reticulum (ER), thereby preventing ER-associated degradation (ERAD) and allowing proper trafficking to the lysosome.14
51 Screening of such libraries on GBA1 might be valuable for the identification of potential chaperones to improve function of impaired GBA1 in Gaucher type 1, but also for identifying GBA1 as an undesired off-target for known drugs and drug-like molecules. This chapter describes the development of an effective FluoPol-ABPP assay for GBA1. The assay is validated by screening the dedicated iminosugar library and returns those library entries already known to inhibit GBA1. The assay is next used to identify GBA1 inhibitors from the Library of Pharmaceutically Active Compounds (LOPAC). This compound collection contains both drugs used in the clinic and compounds that have been studied in clinical trials. Compounds that were identified as GBA1 inhibitors should perhaps be treated with caution: their usage may in a worst-case scenario induce a Gaucher-like phenotype.
3.2 Results and Discussion
GBA1 and GBA2 both are retaining glycosidases employing a catalytic acid-base and a catalytic nucleophile in processing glucosylceramide in a Koshland double displacement mechanism (see Chapter 1). Both enzymes are potently and irreversibly inhibited by the natural product cyclophellitol. As was revealed in Chapter 2, both enzymes react with the cyclophellitol aziridine-derived ABP 1 (ABP
1 depicted in Figure 2 is the same as ABP 14 in Chapter 2), and overexpression of
GBA2 allowed for FluoPol-ABPP screening for compounds acting on GBA2. In the same vein, GBA1 can be brought to overexpression and then assayed with ABP 1. However, in previous years19 it has been shown that GBA1 can also be covalently and irreversibly modified specifically by O6-modified cyclophellitols (O6: glucopyranose numbering and referring to the primary alcohol in cyclophellitol) with the appended moiety containing a BODIPY dye. Based on these findings, ABP
2, containing tetra-aminomethyl rhodamine (TAMRA) as fluorescent tag was
synthesized (scheme 1) as the first objective (the TAMRA dye was chosen based on the results as presented in Chapter 2, in which this dye proved to behave well in FluoPol ABPP assay).
3.2.1 Synthesis and validation GBA1 specific probe
Partially protected cyclohexene 3 was synthesized as described in Chapter 2 and follows the synthetic strategy reported in 2005 by Madsen and co-workers.20 From there the synthetic route towards ABP 2 was undertaken based on methodology described by Witte et al.19 and Li et al.21 The primary alcohol in 3 was converted via selective tosylation and subsequent substitution with an azide into azido alcohol 4. The benzyl groups were removed using BCl3 and all hydroxyl groups were
52
methyl(trifluoromethyl)dioxirane giving a separable mixture of epoxides 6 and 7. Deprotection of 7 with sodium methoxide gave 8-deoxy-8-azidocyclophellitol (8). Azide 8 and TAMRA-alkyne 46 (see for the synthesis of 46 Chapter 2) were conjugated via copper(I)-catalyzed azide-alkyne [2+3] cycloaddition, resulting in the final product, ABP 2 (scheme 1). The two isomers of TAMRA were separated after the click reaction.
Scheme 2: Synthetic route towards ABP 2
Reagents and conditions: Compound 8 was obtained from procedures described in literature by Witte
et al. and Li et al.19,21 (a) i) p-TsCl, Et3N, DCM, 0°C ii) NaN3, DMF, 60°C, 71% (b) i) BCl3, DCM, -78°C, ii) BzCl, pyridine, 70% (c) CF3COCH3, oxone, NaHCO3, MeCN/H2O, 6: 49% 7: 20% (d) NaOMe, MeOH, 75% (e) 5’-TAMRA-alkyne (46), sodium ascorbate, CuSO4, tert-butanol/toluene/H2O (1:1:1 v:v:v), 18h at ambient temperature, 20%.
53 denatured using an excess of sodium dodecylsulfate (SDS), as present in the Laemmli buffer applied, resolved by gel electrophoresis, and the resulting wet gel slabs scanned for fluorescence. As shown in Figure 2B, ABP 1 yields one major band at around 60 kDa – the expected molecular weight for GBA1. Visible as well at much lower intensity are bands at around 100 kDa and 55 kDa and that likely correspond to probe-modified GBA2 and GBA3, respectively. The same major band also appears when using ABP 2. As expected based on the reported highly selective nature of the literature probes on which 2 was modeled, less background labeling was observed, with no discernible modification of either GBA2 or GBA3. Thus and though ABP 1 is on paper suitable for FluoPol ABPP screening for GBA1 inhibitors, ensuing studies were performed with ABP 2.
Figure 2. (A) Reaction mechanism of ABPs 1 and 2 with GBA1. (B) Resulting wet slab from gel-ABPP
labelling of exo-β-glucosidases in mouse brain tissue using 1 µM cyclophellitol aziridine (left) or 1 µM cyclophellitol epoxide (right).
3.2.2 Optimization of FluoPol-ABPP on GBA1
54
reactions were performed in 150 mM McIlvaine buffer supplemented with 0.2% (w/v) sodium taurocholate and 0.1% (v/v) Triton X-100. In order to discriminate
Figure 3. Schematic representation of competitive FluoPol-ABPP in the discovery of GBA1 inhibitors
between enzyme-bound and unbound probe (detected as high and low FluoPol-signal), the reaction conditions were optimized. It was found that the highest polarization signal was obtained when using 5-10 nM probe (Figure 4A) at pH = 5.2 (Figure 4B). The observed optimal pH is consistent with the pH optimum for GBA1.19 Subsequently, three GBA1 inhibitors with varying potency, namely isofagomine (9), N-nonyl-deoxynojirimycin (10) and N-(5-adamantane-1-yl-methoxy-pentyl)-deoxynojirimycin (AMP-DNM, 11) were tested in the assay. The inhibitors were pre-incubated at 37°C with GBA1 for 1 hour before addition of ABP
2. The residual GBA1 activity was determined after overnight probe incubation at
37°C. The competitors showed a dose-dependent response and the potencies of 9,
10 and 11 are respectively 0.23, 8.72 and 4.61 µM (Figure 4C). While the trend in
55
Figure 4. Optimization of (A) probe concentration and (B) pH. (C) Competitive FluoPol ABPP with
inhibitors 9-11. (D) Chemical structures of inhibitors 9 – 11.
3.2.3 Screens of two medium-sized compound libraries
56
Figure 5. (A) Screening of an in-house library containing 358 entries. In total 38 compounds showed
more than 50% inhibition of GBA1. (B) General chemical structures of the two types of iminosugars identified in the screen, with α-aza-C-glycosides 13 and 14 highest GBA1 inhibitory activity. (C) Chemical structures of α-aza-C-glycosides 15 – 20.
These 38 compounds can be divided in two groups; iminosugars containing a biphenyl moiety (12) and two types of α-aza-C-glycosides (one featuring the deoxynojirimycin configuration – 13 and one featuring xylodeoxynojirimycin configuration – 14 – Figure 5B). The iminosugars from group 12 are known nanomolar glucosylceramide synthase (GCS) and GBA2 inhibitors.23 More than 30 of such dual GCS/GBA2 inhibitors have been reported23 to also inhibit GBA1 at micromolar levels. The GBA1 inhibitory potency of α-aza-C-glycosides 13 and 14 – more potent than that of the iminosugars resembling compound 12 – as observed from the competitive FluoPol-ABPP assay (> 90% inhibition) is consistent with the IC50-values reported by Wennekes et al.24
57 screened at a final concentration of 25 µM on GBA1 and the results are presented in Figure 6A.
Figure 6. (A) Screening of the LOPAC library containing 1280 entries at a concentration of 25 µM. (B) +
(C) 17 compounds showed more than 50% GBA1 inhibition. (C) These 6 compounds were validated as true hits with the conventional fluorogenic methylumbelliferyl-β-glucopyranosidesubstrate assay.
The competitive FluoPol ABPP assay yielded 17 compounds (compounds 21 – 37, Figure 6B and 6C) that inhibit GBA1 activity for more than 50% at the final concentration used. These compounds were assessed on their inhibitory potency as well in a fluorogenic substrate assay employing 4-methylumbelliferyl-β-glucopyranoside as the fluorogenic reported substrate. From this orthogonal assay six compounds (the structures of which are depicted in Figure 6C) remained as valid GBA1 inhibitors featuring IC50 values in the low- to sub-micromolar range (see Table
58
positive result for those compounds that fluoresce at the wavelength applied in the assay, (compound 30 for instance structurally closely resembles the TAMRA dye applied in the assay). This may explain why the majority of the 17 compounds do not show up as effective inhibitors in the orthogonal assay. Other compounds such 2-iodoacetamide (37, a generic and highly reactive alkylating agent) comprises known broad-spectrum biological activity and were not evaluated further.
Table 1: IC50 values on GlcCer metabolizing enzymes determined via conventional methods
Inhibitor GBA1a GBA2a GCSb
LO PA C 21* 0.19 13.7 >50 22* 0.20 >1000 >50 (10% inhibition) 23* 1.15 552 >50 24 81.4 >1000 >50 25 34.8 >1000 >50 26* 0.48 151 >50 *Irreversible inhibition, determined via both 4-MU and FluoPol-ABPP assays
a
Inhibition value for in vitro assay is given as IC50 (µM) b Inhibition value for in situ assay is given as IC
50 (µM)
3.2.4 Compounds 21 – 26 are bona fide GBA1 active site binders
59
Figure 7. (A) A schematic representation of ConA washing experiment. (B) Observed GBA1 activities
where [21-26] are 0 – 50 – 250 – 1000 µM.
The activity of GBA1 dropped 85% after exposure of 15 minutes to neutral pH (Figure 7B, 0 µM inhibitor). Compounds 21, 22, 23 and 26 show irreversible inhibition on GBA1 as further decrease of GBA1 activity is observed by increase of their concentration. Compounds 24 and 25 do not show further decrease in GBA1 activity and thus likely act as reversible inhibitors. The apparent Ki and
corresponding binding constant for compounds 21-26 were next determined using a fluorogenic assay with umbelliferyl-β-glucoside as the substrate (Table 2). As shown in Table 2 the binding constants and affinity of the compounds on GBA1 are in accordance with the observed IC50-values reported in Table 1. In addition to 21,
60
Figure 8. Chemical structures of two proton pump inhibitors, rabeprazole (21) and omeprazole (38).
Omeprazole is also present in the LOPAC, but was not able to inhibit GBA1 more than 50% in activity within the used conditions in the screen. As shown in Table 2, omeprazole is able to inhibit GBA1 as well, albeit less potently than its structural analogue, compound 21.
Table 2: Binding constants of LOPAC compounds on GBA1 Inhibitor Ki (min-1) Ki (µM) ki / Ki (µM-1 min-1)
LO PA C 21 0.0113 6.884 0.001642 22 0.00113 4.796 0.000237 23 0.105 12.15 0.008713 24 N.D. 83.6 N.D. 25 N.D. 54.3 N.D. 26 0.076 12.72 0.005998 Omeprazole (38) 0.0831 20.446 0.0041 3.3 Conclusion
61 measured in the assay or because of a known broad-spectrum activity – thus of no biomedical relevance). From follow-up experiments four out of the six compounds turn out to be irreversible inhibitors. The most potent inhibitor of these, rabeprazole (21), is a commercially available proton pump inhibitor (PPI) that suppresses gastroesophageal reflux and is used at high doses as treatment for Zolligner-Ellison syndrome. Omeprazole (38), part of the same PPI family and prescribed as an acid-reflux medicine, proved to be able to inhibit GBA1 as well, though at higher concentrations (omeprazole is present in the LOPAC library as well but did not show up in the initial screen, possibly because the IC50 concentration is
above that of the final concentration in which the LOPAC compounds were assessed). A recent study revealed that PPI’s have a negative impact on the capacity to retain and manipulate spatial memory and planning tasks.25 One theory is that chronic PPI consumption could lead to a malabsorption of vitamin B12 and
results in cognitive decline, however no satisfactory explanation for the observed neurological effects has been found yet. Based on the results described in this chapter, it can be hypothesized that long term use of 21 or 38 might result in Gaucher-like symptoms, including neuropathological manifestations. It should be emphasized that the PPI’s are able to inhibit GBA1 activity, but more research has to be performed in order to proof that these compounds are active site competitors.
3.4 Experimental section
Chemicals, materials and methods
All solvents and reagents were obtained commercially and used as received unless stated otherwise. Dichloromethane (DCM), dimethylformamide (DMF), tetrahydrofuran (THF) and methanol (MeOH) were dried over molecular sieves (4Å/3Å) for at least 12 hours before use. Moisture sensitive reactions were performed under argon atmosphere and carried out in oven-dried glassware. Reactions were monitored by TLC analysis using sheets with pre-coated silica with detection by UV-absorption (254 nm) wherever applicable and by spraying with 20% H2SO4 in MeOH, an aqueous solution containing KMnO4 (5 g/L) and K2CO3 (95
62
acetonitrile, C: 1.0% aqueous trifluoroacetic acid and coupled with an electrospray interface (ESI) was used. For RP-HPLC purifications, an automated HPLC system equipped with a semi-preparative S2 C18 column (5 μm C18, 10Å, 150 × 21.2 mm) was used. The applied buffers were A: H2O + trifluoroacetic acid (1%) and B: MeCN.
HPLC-MS purification was performed on an Agilent Technologies 1200 series automated HPLC system with a Quadropole MS 6130, equipped with a semi-preparative Gemini C18 column (Phenomex, 250 × 10, 5 μm) using buffers A: H2O +
K2CO3 (1%) and B: MeCN. Compounds are characterized by 1H NMR-, 13C NMR-,
63
Scheme 2: Synthesis of 5’-TAMRA-alkyne (46)
Reagents and conditions: (a) NaN3 in DMF, 60°C, 97% (b) PPh3 in 5% HCl (aq), 95% (c) cat. H2SO4 in AcOH under reflux (d) BOP.PF6, DIPEA in DMSO, 12% over two steps
6-Azidohex-1-yne (40)
A mixture of chlorohexyne (0.481 g, 4.1 mmol, 1 eq.) and sodium azide (0.520 g, 8 mmol, 2 eq) in DMF (5 mL) was stirred and heated to 60°C. After 18h the mixture was cooled to room temperature. Subsequently a mixture H2O (25 mL) and diethyl ether (25 mL) was added. The organic layer was
washed with brine (3 × 50 mL), dried with MgSO4 and filtrated. Evaporation of
solvent afforded 35 (0.493 g, 4 mmol, 97%) as a yellow liquid. 1H NMR (300 MHz, CDCl3) 3.33 (t, J = 6.8 Hz, 2H), 2.87 (s, 1H), 2.24 (td, J = 6.7, 2.6 Hz, 2H), 2.15 – 1.85
(m, 2H), 1.85 – 1.53 (m, 2H). 13C NMR (75 MHz, CDCl3) δ 83.7, 68.9, 50.9, 27.8, 25.5,
64
Hex-5-yn-1-amine (41)
Triphenylphosphine(1.06 g, 4 mmol, 1 eq.) dissolved in THF (20 mL) was dropwisely added into a solution of 35 (0.493 g, 4 mmol) in 5% aqueous HCl (5 mL). Addition was performed in 30 minutes at room temperature and the reaction was stirred for an additional 1.5 hours. The phases were separated after addition of 50 mL H2O using a separation funnel and the
aqueous layer was washed using diethyl ether (3 × 75 mL). Then the pH of the aqueous layer was adjusted to pH=10 using KOH pellets. Product was extracted with DCM (3 × 50 mL). Combined organic layers were dried over MgSO4 and
filtered. After removal of the organic solvents under reduced pressure yellow oil (0.37 g, 3.8 mmol, 95%) was afforded. 1H NMR (300 MHz, CDCl3) 2.89 (s, 1H), 2.69
(t, J = 6.8 Hz, 2H),2.42 (td, J = 6.7, 2.6 Hz, 2H), 1.85 – 1.53 (m, 4H), 1.42 (s, 2H). 13C NMR (75 MHz, CDCl3) δ 83.9, 68.8, 41.1, 31.5, 25.6, 18.1. HRMS: found 98.0965 [M+H]+, calculated for [C6H11N1+H]+ 98.0964 5-carboxy-2-(6-(dimethylamino)-3-(dimethyliminio)-3H-xanthen-9-yl) benzoate(44&45) Dimethylaminophenol (42, 6.9 g, 50 mmol) and trimellitic anhydride (43, 4.8 g, 25 mmol) were dissolved in AcOH (400 mL). After adding a catalytic amounts of concentrated H2SO4 (ca. 0.5 mL) the mixture was refluxed overnight. Reaction mixture was
concentrated under reduced pressure and pre-purified over column chromatography (DCM 50% MeOH/DCM) to isolate 2.7 g of a mixture containing desired of regio-isomers.
2-(6-(dimethylamino)-3-(dimethyliminio)-3H-xanthen-9-yl)-5-(hex-5-yn-1-yl carbamoyl)benzoate (46)
BOP.PF6 (730 mg, 1.65 mmol, 1.1 eq) and DIPEA (500 µL, 2.87 mmol, 1.9 eq) were added into a mixture containing isomers 44 and 45 (657 mg, 1.53 mmol, 1 eq) and linker 41 (150 mg, 1.54 mmol, 1 eq) dissolved in DMSO (25 mL). The reaction was stirred for 24 hours at ambient temperature. Desired stereoisomeric product 46 (94 mg, 184 µmol, 12%
estimated yield over two steps) was isolated using HPLC purification. 1H NMR (400
65 3.52 (t, J = 6.9 Hz, 2H), 2.35 – 2.21 (m, 3H), 1.90 – 1.72 (m, 2H), 1.67 (dq, J = 9.9, 7.0 Hz, 2H), 1.32 (s, 1H). 13C NMR (101 MHz, MeOD) δ 166.76, 165.97, 159.33, 157.68, 157.60, 136.67, 136.43, 131.46, 113.35, 68.44, 48.24, 48.03, 47.81, 47.60, 47.39, 47.18, 46.96, 39.28, 28.17, 25.74, 17.37. HRMS: found 510.2384 [M+H]+, calculated for [C31H31O4N3+H]+ 510.2387 (1R,2R,5S,6S)-2-(azidomethyl)-5,6-bis(benzyloxy)cyclohex-3-enol (4)
To a solution of 3 (1.24 g, 3.65 mmol) in DCM (26 mL) were added p-toluenesulfonylchloride (1.04 g, 5.48 mmol, 1.1 eq.) and triethylamine (0.90 mL, 6.57 mmol, 1.8 eq.) at 0°C. The solution was stirred for 5 h before being poured in 1M HCl solution. The mixture was extracted with Et2O and the organic layer was dried over MgSO4 before being concentrated in
vacuo. The residue was dissolved in DMF (35 ml) and sodium azide (2.40 g, 36.7
mmol, 10.4 eq.) was added. The solution was stirred for 24 h at 60°C before being concentrated in vacuo. The crude product was diluted with EtOAc, washed with 1 M HCl, saturated aqueous NaHCO3 and brine. The combined organic layers were
dried over MgSO4 and concentrated in vacuo. Purification by silica column
chromatography (8%→16% EtOAc in petroleum ether) afforded 4 (900 mg, 2.46 mmol, 71%) as an amorphous solid. 1H NMR (400 MHz, CDCl3): δ 7.33-7.26 (m,
10H), 5.79 (dt, J = 10.4, 2.4 Hz, 1H), 5.58 (dt, J = 10.4, 2.4 Hz, 1H), 5.02 (d, J = 11.3, 1H), 4.7 (dd, J = 11.2, 5.4 Hz, 2H), 4.65 (d, J = 11.2 Hz, 1H), 4.21-4.19 (m, 1H), 3.61-3.53 (m, 3H), 3.44 (dd, J = 12.0, 6.0 Hz, 1H), 2.83 (s, 1H) and 2.48 (br, 1H). 13C NMR (100 MHz, CDCl3) δ 138.1, 137.2, 128.7, 128.6, 128.0, 127.9, 127.8, 127.7, 83.5, 80.3, 75.0, 71.6, 52.5 and 43.6. HRMS found 366.1813 [M+H]+, calculated for [C21H23N3O3+H]+ 366.1814.
(1R,2R,3S,6R)-6-(azidomethyl)cyclohex-4-ene-1,2,3-triyl tribenzoate (5)
Borontrichloride (21 mL, 1M in DCM, 21.1 mmol, 10 eq.) was added to a solution of 11 (777.1 mg, 2.11 mmol) in anhydrous DCM (10 mL) at -78°C. The reaction mixture was stirred at -78°C for 6h before being quenched with MeOH. The solution was concentrated in vacuo giving the triol intermediate, which was immediately used for benzoylation. The crude product was coevaporated several times with anhydrous toluene before being dissolved in pyridine (10 mL). Benzoyl chloride (2.6 mL, 21.1 mmol, 10 eq.) was added at 0°C and the reaction mixture was stirred for 18h at ambient temperature. The mixture was quenched with saturated aqueous NaHCO3, extracted with EtOAc,
dried over MgSO4 and concentrated in vacuo. Purification by silica column
66
mmol, 70%) as yellow oil. 1H NMR (400 MHz, CDCl
3) δ 7.99 (d, J = 7.2 Hz, 2H), 7.92 (d, J = 7.2 Hz, 2H), 7.84 (d, J = 7.2 Hz, 2H), 7.53-7.46 (m, 3H), 7.40 (dt, J = 24.4, 8.0 Hz, 5H), 7.26-7.18 (m, 2H), 6.00-5.93 (m, 3H), 5.86 (d, J = 10.0 Hz, 1H), 5.72 (t, J = 9.2 Hz, 1H), 3.64 (dd, J = 12.4, 4.0 Hz, 1H), 3.46 (dd, J = 12.4, 6.4 Hz, 1H) and 2.99-2.97 (m, 1H). 13C NMR (100 MHz, CDCl3) δ 166.0, 165.9, 133.3, 133.2, 133.1, 129.8, 129.7, 129.6, 129.4, 129.0, 128.9, 128.5, 128.4, 128.3, 126.2, 127.0, 72.7, 72.7, 72.6, 70.4, 52.0 and 42.5. HRMS found 498.1660 [M+H]+, calculated for [C28H23N3O6+H]+ 498.1662.
(1S,2S,3S,4R,5S,6S)-5-(azidomethyl)-7-oxabicyclo[4.1.0]heptane-2,3,4-triyl tri-
benzoate (6) and (1R,2S,3S,4R,5S,6R)-5-(azidomethyl)-7-oxabicyclo[4.1.0]
heptane-2,3,4-triyl tribenzoate (7)
A solution of 0.4 mM Na2EDTA solution in H2O (3.1 mL) and trifluoroacetone (1.34
mL, 15 mmol, 15 eq.) were added to 5 (497 mg, 1.0 mmol) in acetonitrile (6.7 mL). A mixture of oxone (3.07 g, 5.0 mmol, 5 eq.) and NaHCO3 (588.1 mg, 7.0 mmol, 7
eq.) was added to the solution over a period of 15 minutes. After stirring at 4°C for 4h, an additional amount of 0.4 mM Na2EDTA in H2O (1.5 mL), trifluoroacetone (0.7
mL, 7.5 mmol, 7.5 eq.) and a mixture of oxone (1.5 g, 2.5 mmol, 2.5 eq.) and NaHCO3 (290 mg, 3.5 mmol, 3.5 eq.) were added to the reaction mixture over a
period of 15 min. The reaction mixture was stirred at 4°C for 30 minutes before being diluted with H2O. After extraction of the water layer with EtOAc, the
combined organic layers were dried over MgSO4 and concentrated in vacuo.
Purification by silica column chromatography (8%→10% Et2O in petroleum ether) and (16%→18% Et2O in petroleum ether) afforded 6 (103.9 mg, 0.20 mmol, 20%)
and 7 (253.7 mg, 0.49 mmol, 49%) respectively as amorphous solid.
6: 1H NMR (400 MHz, CDCl3) δ 8.02 (d, J = 7.2 Hz, 2H), 7.89 (d, J = 7.2 Hz, 2H), 7.78 (d, J = 7.2 Hz, 2H), 7.53-7.19 (m, 5H), 5.96 (t, J = 9.6 Hz, 1H), 5.77 (d, J = 8.8 Hz, 1H), 5.55 (t, J = 9.6 Hz, 1H), 3.773.74 (m, 2H), 3.64 (dd, J = 12.8, 4.0 Hz, 1H), 3.32 (s, 1H) and 2.68 (ddd, J = 9.2, 5.2, 3.8 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 166.0, 165.9, 165.6, 133.4, 133.0, 129.9, 129.7, 129.5, 129.0, 128.9, 128.6, 128.4, 128.3, 128.1, 72.1, 70.0, 69.9, 54.6, 53.8, 50.8 and 40.9. HRMS found 514.1602 [M+H]+, calculated for [C28H23N3O7+H]+ 514.1609.
67 128.4, 128.3, 128.1, 72.2, 71.4, 67.8, 54.7, 54.2, 50.5 and 40.8. HRMS found 514.1609 [M+H]+, calculated for [C28H23N3O7+H]+ 514.1609.
(1S,2R,3S,4R,5R,6R)-5-(azidomethyl)-7-oxabicyclo[4.1.0]heptane-2,3,4-triol (8)
A catalytic amount of NaOMe was added to a solution of 7 (103.9 mg, 0.20 mmol) in MeOH (1.0 mL) and stirred for 1h at ambient temperature. The reaction mixture was neutralized with Amberlite IR-120H+, filtered and concentrated in vacuo. Purification by silica column chromatography (6%→8% MeOH in DCM) provided 8 (30.0 mg, 0.15 mmol, 75%).
1 H NMR (400 MHz, MeOD) δ 3.84 (dd, J = 8.4, 3.6 Hz, 1H), 3.67 (d, J = 8.0 Hz, 1H), 3.51 (dd, J = 12.0, 8.8 Hz, 1H), 3.36 (d, J = 3.2 Hz, 1H), 3.23 (dd, J = 10.0, 8.4 Hz, 1H), 3.13-3.08 (m, 2H), 2.07 (ddt, J = 9.4, 3.6, 1.6 Hz, 1H). 13C NMR (100 MHz, MeOD) δ 78.3, 72.7, 68.6, 57.6, 56.1, 52.4 and 43.9. HRMS found 202.0825 [M+H]+, calculated for [C7H11N3O4+H]+ 202.0822. 2-(6-(dimethylamino)-3-(dimethyliminio)-3H-xanthen-9-yl)-5-((4-(1-(((1R,2R,3R,4S, 5R,6S)-3,4,5-trihydroxy-7-oxabicyclo[4.1.0]heptan-2-yl)methyl)-1H-1,2,3-triazol-4-yl)butyl)carbamoyl)benzoate (ABP 2)
Compound 8 (6 mg, 0.03 mmol, 1 eq) and 46 (15.2 mg, 0.03 mmol, 1 eq) were dissolved in tert-butanol/toluene/H2O (3 mL, 1:1:1, v/v/v). CuSO4
(0.06 mL, 0.1 M in H2O) and sodium ascorbate
(0.06 mL, 0.1 M in H2O) were added and the
reaction mixture was stirred at room temperature for 18h under argon atmosphere. Then, the solution was diluted with CH2Cl2, washed with H2O, dried over MgSO4 and
concentrated under reduced pressure. The crude was purified by silica gel column chromatography (CH2Cl2 to CH2Cl2/MeOH 9:1), subsequently purified by
semipreparative reversed-phase HPLC (linear gradient: 24% to 28% B in A, 12 min, solutions used A: 50mM NH4HCO3 in H2O, B: MeCN) and lyophilized to yield ABP 2
68
114.8, 97.3, 78.3, 72.6, 68.7, 57.6, 55.6, 50.7, 49.6, 49.5, 49.4, 49.3, 49.2, 49.0, 48.9, 48.7, 48.6, 44.7, 40.8, 40.7, 29.8, 27.8, 25.9. HRMS: found 711.3140 [M+H]+, calculated for [C38H43O8N6+H]+ 711.3142.
Gel-Activity Based Protein Profiling experiments on tissues
Lysates of mouse brain tissue were prepared by osmolysis of the tissue in McIlavine buffer (pH = 5.0), supplemented with 0.25M sucrose, 0.2% sodium taurocholate (w/v) and 0.1% Triton X-100 (v/v). The lysates were homogenized using sonication, after which the total protein concentration was determined via a Bradford assay, using BSA (Sigma) for standards and BioRad Quickstart Bradford Reagents. Samples (Vfinal = 20 µL) containing 10 µg protein were incubated for 30 minutes at 37°C with
1 µM probe (1 or 2). Protein content was denatured using Laemmli Buffer (4x) at 100°C for 3 minutes. Reactions were resolved by 12.5% SDS-PAGE electrophoresis and wet slabs were scanned for fluorescence (Molecular Imager Gel Doc XR, Biorad).
Optimization of the FluoPol-ABPP assay
Pure recombinant human enzyme (Cerezyme from Genzyme) was used as GBA1 protein source. The optimal probe concentration on FluoPol signal was determined by varying probe concentrations from 1 nM to 50 nM probe at a constant protein concentration (2 µg/mL) and at pH = 5.2. FluoPol-ABPP assays were also performed at different pH values by preparation of different McIlvaine buffers. These pH-experiments were performed at optimal probe concentration (5 nM). Competition experiments were conducted by 1 hour pre-incubation of compounds in the protein solution at 37°C (2.5% DMSO). All reactions (Vfinal = 25 µL) were
supplemented with 0.2% sodium taurocholate (w/v), 0.1% Triton X-100 (v/v) and were carried out in 384-wells plates (small-volume black, Greiner). FluoPol-signals were monitored on an Infinite M1000Pro (Tecan) using λex 530 nm and λem 580 nm.
Samples containing an excess of isofagomine were used as reference samples (0% probe labelling), samples without inhibitors for 100% labelling controls and samples without probe as blanks to correct for background polarization. All samples were corrected for background polarization and the residual enzyme activity was calculated based on the polarization signal from the controls. Polarization signals were plotted against time or inhibitor concentration and processed in GraphPad Prism 6.0. IC50 values were calculated via non-linear regression using mentioned
software (N=2, n=3).
FluoPol-ABPP screen of the LOPAC and iminosugar libraries
69 in 384-well black-bottom plates (Greiner) with final reaction volumes of 15 µL. Concentration of the LOPAC and iminosugar library were respectively 25 and 5 µM. The FluoPol signal was measured on a ClarioStar (BMG Labtech). Resulting polarization signals were processed as described above. Residual enzyme activities were plotted against the corresponding compound ID.
Analysis of selected compounds as inhibitors of enzymatic activity of GCS, GBA1 and GBA2
All IC50 values were determined in triplicate and the inhibitors tested were
pre-incubated for 30 minutes at 37°C. Observed fluorescence was curve-fitted against inhibitor or substrate concentrations using GraphPad Prism 6.0 in order to obtain the IC50 values.
GBA1: Pure recombinant human enzyme (Cerezyme from Genzyme) was used.
Activity was measured with 3.7 mM 4-methylumbeliferone (4MU)-β-D-glucopyranoside (Sigma) in 150 mM McIlvaine buffer pH 5.2 supplemented with 0.2% sodium taurocholate (w/v), 0.1% Triton X-100 (v/v), 0.1% bovine serum albumin (BSA) (w/v) for 30 min. The reaction was stopped with excess 1M NaOH-Glycine (pH 10.3), liberated 4MU fluorescence was measured with a fluorimeter LS55 (Perkin Elmer) using λEx 366 nm and λEm 445 nm.
GBA2: For GBA2 measurements, cellular homogenates of a stable HEK293T
over-expressing GBA2 cell line pre-incubated for 30 min with an inhibitor of GBA1 (1 mM conduritol β epoxide CBE from Sigma) were used. Activity was measured with 3.7 mM 4MU-β-D-glucopyranoside in 150 mM McIlvaine pH 5.8, 0.1% BSA (w/v) for 1 h. Reactions were stopped as described above for GBA1.
GCS: IC50 values for GCS were determined in vivo with 6-[N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-ylaminododecanoyl]sphingosine (NBD-ceramide) as substrate. RAW 264.7 cells were grown to confluence in 6-well plates and pre-incubated for 1h with an inhibitor of GBA1 activity (300 µM CBE), followed by 1h incubation at 37°C with 1 µM C6-NBD-ceramide and in the presence of a range of inhibitor concentrations. The cells were washed 3x with PBS and harvested by scraping. After lipid extraction (described by Bligh and Dyer),26 the C6-NBD lipids were separated and detected by HPLC (λEx 470 nm and λEm 530 nm). IC50 values were
determined in duplicate from the titration curves of observed formed C6-NBD-glucosylceramide and data was curve-fitted via GraphPad Prism 6.0.
Determination of binding constants for GBA127
70
slopes were calculated with Graphpad Prism 6.0 using linear regression. A plot of k’ against [I] fitted to the hyperbolic equation k’ = (kinact [I]/Ki + [I]) was used to
determine Ki (equilibrium constant) and ki (rate constant), calculated using
non-linear regression function within Graphpad Prism 6.0. Inhibition constants for reversible inhibitors were determined by the method described in Chapter 2. Concanavalin A experiment
Cerezyme (Genzyme) was diluted 1000x in 150 mM McIlvaine buffer pH 5.2 supplemented with 0.2% sodium taurocholate (w/v), 0.1% Triton X-100 (v/v), 0.1% bovine serum albumin (BSA) (w/v) and competitors were added in concentrations varying from 0 to 1 mM, except for the controls, and incubated at 37°C for 15 minutes at pH 5.2. pH was brought to 7 by adding DMEM high glucose (Sigma) supplemented with 10% NBS and 100 units/mL penicillin/streptomycin (Gibco) and the samples were incubated for 15 minutes at 37°C. Then a small quantity of the ConA beads were added to each sample and all samples were incubated at 4°C for 1 hour. The samples were then washed three times with a 0.1 M sodium acetate solution (pH = 6.0) containing 0.1 M NaCl, 1 mM CaCl2 and 1 mM MnCl2. The
residual GBA1 activity was measured via the fluorogenic 4-MU assay as described above. Experiments were performed in triplicate.
3.5 References
(1) Wennekes, T., van den Berg, R. J. B. H. N., Boot, R. G., van der Marel, G. A., Overkleeft, H. S., and Aerts, J. M. F. G. (2009) Glycosphingolipids - nature, function, and pharmacological modulation. Angew. Chem. Int. Ed. 48, 8848–8869.
(2) Lombard, V., Golaconda Ramulu, H., Drula, E., Coutinho, P. M., and Henrissat, B. (2014) The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids
Res. 42, D490–D495.
(3) Boot, R. G., Verhoek, M., Donker-Koopman, W., Strijland, A., van Marle, J., Overkleeft, H. S., Wennekes, T., and Aerts, J. M. F. G. (2007) Identification of the non-lysosomal glucosylceramidase as β-glucosidase 2. J. Biol. Chem. 282, 1305– 1312.
(4) Körschen, H. G., Yildiz, Y., Raju, D. N., Schonauer, S., Bönigk, W., Jansen, V., Kremmer, E., Kaupp, U. B., and Wachten, D. (2013) The Non-lysosomal β-Glucosidase GBA2 Is a Non-integral Membrane-associated Protein at the Endoplasmic Reticulum (ER) and Golgi. J. Biol. Chem. 288, 3381–3393.
(5) Brady, R. O., Kanfer, J. N., Bradley, R. M., and Shapiro, D. (1966) Demonstration of a deficiency of glucocerebroside-cleaving enzyme in Gaucher’s disease. J. Clin.
71
(6) Patrick, A. D. (1965) A deficiency of glucocerebrosidase in Gaucher’s disease.
Biochem. J. 97, 17C-24C.
(7) Beutler, E. (1988) Gaucher disease. Blood Rev. 2, 59–70.
(8) Cox, T. M. (2001) Gaucher disease: understanding the molecular pathogenesis of sphingolipidoses. J. Inherit. Metab. Dis. 24 Suppl 2, 106–21; discussion 87-88. (9) Goker-Alpan, O., Lopez, G., Vithayathil, J., Davis, J., Hallett, M., and Sidransky, E.
(2008) The spectrum of Parkinsonian manifestations associated with glucocerebrosidase mutations. Arch. Neurol. 65, 1353–1357.
(10) Aflaki, E., Westbroek, W., and Sidransky, E. (2017) The complicated relationship between Gaucher disease and Parkinsonism: insights from a rare disease. Neuron
93, 737–746.
(11) Grabowski, G. A. (2008) Phenotype, diagnosis, and treatment of Gaucher’s disease.
Lancet 372, 1263–1271.
(12) Platt, F. M. (1997) Prevention of lysosomal storage in Tay-Sachs mice treated with N-butyldeoxynojirimycin. Science 276, 428–431.
(13) Aerts, J. M., Hollak, C., Boot, R., and Groener, A. (2003) Biochemistry of glycosphingolipid storage disorders: implications for therapeutic intervention.
Philos. Trans. R. Soc. Lond. B. Biol. Sci. (Dwek, R. A., Butters, T. D., Platt, F. M., and
Cox, T. M., Eds.) 358, 905–914.
(14) Boyd, R. E., Lee, G., Rybczynski, P., Benjamin, E. R., Khanna, R., Wustman, B. A., and Valenzano, K. J. (2013) Pharmacological chaperones as therapeutics for lysosomal storage diseases. J. Med. Chem. 56, 2705–2725.
(15) Lahav, D., Liu, B., van den Berg, R. J. B. H. N., van den Nieuwendijk, A. M. C. H., Wennekes, T., Ghisaidoobe, A. T., Breen, I., Ferraz, M. J., Kuo, C.-L., Wu, L., Geurink, P. P., Ovaa, H., van der Marel, G. A., van der Stelt, M., Boot, R. G., Davies, G. J., Aerts, J. M. F. G., and Overkleeft, H. S. (2017) A fluorescence polarization activity-based protein profiling assay in the discovery of potent, selective inhibitors for human nonlysosomal glucosylceramidase. J. Am. Chem. Soc. 139, 14192– 14197.
(16) Witte, M. D., Walvoort, M. T. C., Li, K.-Y., Kallemeijn, W. W., Donker-Koopman, W. E., Boot, R. G., Aerts, J. M. F. G., Codée, J. D. C., van der Marel, G. A., and Overkleeft, H. S. (2011) Activity-based profiling of retaining β-glucosidases: a comparative study. ChemBioChem 12, 1263–1269.
72
(18) Witte, M. D., van der Marel, G. a, Aerts, J. M. F. G., and Overkleeft, H. S. (2011) Irreversible inhibitors and activity-based probes as research tools in chemical glycobiology. Org. Biomol. Chem. 9, 5908-5926.
(19) Witte, M. D., Kallemeijn, W. W., Aten, J., Li, K.-Y., Strijland, A., Donker-Koopman, W. E., van den Nieuwendijk, A. M. C. H., Bleijlevens, B., Kramer, G., Florea, B. I., Hooibrink, B., Hollak, C. E. M., Ottenhoff, R., Boot, R. G., van der Marel, G. a, Overkleeft, H. S., and Aerts, J. M. F. G. (2010) Ultrasensitive in situ visualization of active glucocerebrosidase molecules. Nat. Chem. Biol. 6, 907–913.
(20) Hansen, F. G., Bundgaard, E., and Madsen, R. (2005) A short synthesis of (+)-cyclophellitol. J. Org. Chem. 70, 10139–10142.
(21) Li, K.-Y., Jiang, J., Witte, M. D., Kallemeijn, W. W., van den Elst, H., Wong, C.-S., Chander, S. D., Hoogendoorn, S., Beenakker, T. J. M., Codée, J. D. C., Aerts, J. M. F. G., van der Marel, G. A., and Overkleeft, H. S. (2014) Synthesis of cyclophellitol, cyclophellitol aziridine, and their tagged derivatives. Eur. J. Org. Chem. 2014, 6030– 6043.
(22) Ghisaidoobe, A., Bikker, P., de Bruijn, A. C. J., Godschalk, F. D., Rogaar, E., Guijt, M. C., Hagens, P., Halma, J. M., van’t Hart, S. M., Luitjens, S. B., van Rixel, V. H. S., Wijzenbroek, M., Zweegers, T., Donker-Koopman, W. E., Strijland, A., Boot, R., van der Marel, G., Overkleeft, H. S., Aerts, J. M. F. G., and van den Berg, R. J. B. H. N. (2011) Identification of potent and selective glucosylceramide synthase inhibitors from a library of N-alkylated iminosugars. ACS Med. Chem. Lett. 2, 119–123. (23) Ghisaidoobe, A. T., van den Berg, R. J. B. H. N., Butt, S. S., Strijland, A.,
Donker-Koopman, W. E., Scheij, S., van den Nieuwendijk, A. M. C. H., Koomen, G.-J., van Loevezijn, A., Leemhuis, M., Wennekes, T., van der Stelt, M., van der Marel, G. A., van Boeckel, C. A. A., Aerts, J. M. F. G., and Overkleeft, H. S. (2014) Identification and development of biphenyl substituted iminosugars as improved dual glucosylceramide synthase/neutral glucosylceramidase inhibitors. J. Med. Chem.
57, 9096–9104.
(24) Wennekes, T., van den Berg, R. J. B. H. N., Boltje, T. J., Donker-Koopman, W. E., Kuijper, B., van der Marel, G. A., Strijland, A., Verhagen, C. P., Aerts, J. M. F. G., and Overkleeft, H. S. (2010) Synthesis and evaluation of lipophilic aza-C-glycosides as inhibitors of glucosylceramide metabolism. Eur. J. Org. Chem. 2010, 1258–1283. (25) Akter, S., Hassan, M. R., Shahriar, M., Akter, N., Abbas, M. G., and Bhuiyan, M. A.
(2015) Cognitive impact after short-term exposure to different proton pump inhibitors: assessment using CANTAB software. Alzheimers. Res. Ther. 7, 79. (26) Bligh, E. G., and Dyer, W. J. (1959) A rapid method of total lipid extraction and
purification. Can. J. Biochem. Physiol. 37, 911–917.
