The handle
http://hdl.handle.net/1887/87273
holds various files of this Leiden University
dissertation.
Author: Lahav, D.
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4
A Fluorescence Polarization Activity Based Protein Profiling Assay on
Human Lysosomal Acid α-Glucosidase for the Identification of Competitive
Inhibitors
4.1 Introduction
Lysosomal acid α-glucosidase (GAA, a GH31 glycohydrolase) degrades glycogen by employing an acid/base catalyzed double displacement mechanism (Figure 1).1-3 The most likely conformational itinerary of the substrate during hydrolysis is 4C1 4
H3 1S3, which is the reversed order compared to most retaining β-glucosidases.4,19 Deficiency in GAA leads to accumulation of glycogen in lysosomes, resulting in the lysosomal storage disorder called Pompe disease (also known as glycogen storage disease type 2).5,6 Glycogen accumulation leads to cellular damage in all tissues, and in particular in cardiac and skeletal muscle.
Figure 1. Lysosomal degradation of glycogen by GAA, where D518 acts as the nucleophilic residue and D616 as the acid/base.7,8 The enzyme hydrolyzes at the terminal non-reducing end of the substrate.
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through intravenous administration of recombinant human GAA (rhGAA). ERT for Pompe disease was approved in 2006.9,10 The efficiency of the treatment is limited due to insufficient targeting and uptake in muscle tissues and by immunogenic reactions.11-13 Studies indicate that early treatment, that is, before extensive muscular damage has occurred, can lead to improvement in motor and respiratory function.9 It is clear that alternative therapeutic strategies for the treatment of Pompe disease are needed.
It has been proposed that small-molecule pharmacological chaperones co-administered with rhGAA may yield effective treatments for patients suffering from Pompe disease.14 For example deoxynojirimycin (DNM; 1, Figure 2A) that stabilizes rhGAA through reversible active site occupancy, extends rhGAA circulation time, enhances its bio-distribution and thus increases the effectiveness of ERT.14,15
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Figure 2. (A) Chemical structures of reported GAA chaperones deoxynojirimycin (1) and N-acetylcysteine (2). (B) Binding mechanism of an α-configured cyclophellitol-aziridine probe on GAA.
It was rationalized that fluorescence polarization activity-based protein profiling (FluoPol-ABPP) could be a useful tool for the identification of GAA inhibitors. The work described by Jiang et al.17 shows that labeling of active GAA is possible using 1,6-epi-cyclophellitol aziridine (the cyclophellitol aziridine derivative featuring the configuration of α-glucopyranose), the aziridine nitrogen of which is functionalized to contain a fluorophore (for in-gel fluorescence detection) or a biotin (for mass spectrometry-based detection and analysis of reacted protein). In order to stay in tune with the other chapters, an α-configured aziridine probe grafted with tetra-aminomethylrhodamine (TAMRA) as fluorescent tag was synthesized. The synthesis of this probe and its use in the competitive FluoPol ABPP-mediated discovery of GAA inhibitors from the in-house iminosugar compound library (see for the complete list of these compounds the Appendix at the end of this Thesis) is described here.
4.2 Results and Discussion
The assays described in the previous chapters are based on activity-based probes (ABPs) that are able to form a covalent linkage within accessible active sites of the targeted exo-glycosidases. Following on probe synthesis and target validation, the conditions for FluoPol-ABPP were optimized and the assays were validated using the iminosugar-based library. The results from these preceding chapters were taken into consideration in the design of the experiments, the outcome of which is detailed below.
4.2.1 Synthesis and validation of a FluoPol compatible GAA probe
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(3) was synthesized from D-xylose following the procedures described in Chapter 2 and based on the procedure originally developed by Hansen et al.18 Installation of the α-configured aziridine and subsequent N-alkylation was performed according to the synthesis procedure described by Jiang et al.17 In brief, 3 was debenzylated using Birch conditions and a benzylidene protective group was introduced to mask the 4-6-dihydroxyl moiety (glucopyranose numbering) yielding compound 5. Exposure of 5 to trichloroacetonitrile in the presence of DBU gave imidate 6. Subsequent iodocyclization was realized by adding iodine (7) and once complete (after one day) was followed by subsequently adding acid (to hydrolyze the in situ formed iminal) and excess sodium bicarbonate (to deprotonate the in situ formed ammonium salt and effect intramolecular cyclisation) to the reaction mixture, forming aziridine 8. The nitrogen in compound 8 was alkylated with iodo-azido-octane resulting in compound 9, and ensuing copper(I)-catalyzed click reaction of the azide in 9 with the alkyne in TAMRA derivative 34 afforded GAA ABP 10.
Scheme 3: Synthetic route towards ABP 10
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MeOH, yield over two steps 63% (f) 1-azido-8-iodooctane, K2CO3, DMF, 80°C, 39% (g) Mixture of 5’/6’-TAMRA (34), sodium ascorbate, CuSO4 in DMF, 3 days at ambient temperature, 21%.
ABP 10 was used in an in vitro ABPP experiment on mouse brain tissue. In this experiment ABP 10 was compared for its labelling capability with two established ABPs that were synthesized as described by Jiang et al.17 (ABPs 11 and 12). These α-configured aziridines, depicted in Figure 3A, were incubated with 10 µg mouse brain tissue for 30 minutes at 37°C. The reactions were quenched with Laemmli buffer, which contains an excess of sodium dodecylsulfate (SDS), and the protein mixtures (which were denatured as a result of the quench with the Laemmli buffer) resolved via gel-electrophoresis. The formed covalent enzyme-ABP adducts were visualized after fluorescence scanning of the wet gel slabs. As shown in Figure 3B, the tested probes are able to label mouse homologues of GAA and ER-α-glucosidase II (GANAB) at acidic pH (pH = 5.0).
Figure 3. (A) Chemical structures of cyclophellitol-based probes 9 - 11. (B) Gel-ABPP experiment using 1 µM probe 10, 11 or 12 labelling GANAB and GAA in mouse brain tissue at acidic pH.
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4.2.2 Optimization & Validation of FluoPol-ABPP on GAA
The drug that is used in ERT for the treatment of Pompe disease is called Myozyme®, the active ingredient of which comprises recombinant human GAA. Myozyme was used in the FluoPol-ABPP experiments in order to ensure binding specificity between probe and enzyme (as an alternative and in line with the approach taken in Chapter 2, GAA could in principle be brought to overexpression after which crude cell extracts could be used in FluoPol ABPP assays).
A schematic representation of such a FluoPol-ABPP assay is depicted in Figure 4 – this exact scheme has also been shown in the previous chapter, however here the blue moiety resembled GAA and the green-black-pink star part ABP 10. The reaction conditions for probe labelling on rhGAA were optimized in a 384-well format (Vfinal = 25 µL). The biochemical reactions were conducted with 10 µg/mL Myozyme in 150 mM McIlvaine buffer (0.1% BGG; 0.5 mg/mL Chaps). An optimal FluoPol signal was obtained when using 25 nM probe and at pH = 5.0 as shown in respectively Figures 5A and 5B. Pre-incubation of established inhibitors such as 1 (Ki = 110 nM)16, a known reversible GAA inhibitor, or cyclophellitol α-cyclosulfate (13) (IC50 = 82 nM)19, a known irreversible GAA inhibitor, gave a dose-dependent inhibition (Figure 5C and 5D). From this assay it appears that compound 1 is a low micromolar GAA inhibitor, and 13 nanomolar potent on GAA. These observed potencies are consistent with the literature values reported by Artola et al.19, validating the FluoPol-assay.
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Figure 5. Optimization of (A) probe concentration and (B) pH for FluoPol-assay. (C) Dose-dependent response of known GAA inhibitors and (D) observed inhibitory potency of 1 and 13 (determined via FluoPol-ABPP).
4.2.3 Screen of an iminosugar-based compound library on rhGAA
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Figure 6. (A) Screening of an in-house library containing 358 entries at a concentration of 5 µM. Approximately 80 compounds showed more than 50% inhibition on GAA. (B) Representative members of three categories of compounds that show more than 50% inhibition on GAA in the FluoPol-assay and can be categorized into three groups; iminosugars containing aromatic groups (14 – 16), dialkyl ether-substituted iminosugars (17 –19) and α-aza-C-glycosides (20 – 21).
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Table 1: IC50 values (in µM) of iminosugars on GAA and GANAB determined via fluorogenic methods, which are described in the experimental section. Shown as well are calculated GANAB/GAA ratios, with a stronger shade of green representing higher GAA selectivity.
82 150 14.7 1.375 12.73 9.262 343 0.6 0.043 0.25 5.798 153 13.8 3.649 19.39 5.313 345 1.5 0.058 0.37 6.465 154 2.4 8.797 21.93 2.493 347 0.4 0.042 0.40 9.472 158 28.9 2.462 100.00 40.617 348 3.1 0.023 0.61 26.462 159 22.5 0.449 100.00 222.519 349 3.0 0.028 4.35 155.874 170 4.1 0.428 1.62 3.780
The number of iminosugars interacting with GAA and GBA2 is larger than that interacting with GAA and GBA1, and with GBA1 and GBA2. In total 76 iminosugars act on GAA and GBA2, and of these 38 also inhibit GBA1. The five iminosugars that were exclusively picked up in the GAA screen, are depicted in Figure 7. Compounds
22 – 26, corresponding with iminosugars with ID’s (Appendix) #66, 89, 100, 154 and
200, have not emerged in the GBA1/GBA2 screens described in the previous chapters, even though the configuration of the iminosugar (glucose/galactose) matches that of many GBA1/GBA2 inhibitors known and that do show up in the assays described in the previous chapters. This does not necessarily mean that compounds 22-26 do not inhibit the glucosylceramidase hydrolyzing enzymes, but it does imply that they do so less effectively than other compounds (at least under the assay conditions used). Interestingly, compound 22 features a branched apolar fluorenylmethyl group. Such groups, and in contrast to linear apolar groups appended to deoxynojirimycins, are known to be poor inhibitors of GBA1 and GBA220 and it might be worthwhile to explore this design in more depth with the aim to identify selective GAA inhibitors.
Figure 7. Chemical structures of five hits that emerged exclusively in the GAA screen subject of this chapter, and not in the GBA1/GBA2 screens of the two preceding chapters.
4.3 Conclusion
83 5.0. The secondary, orthogonal, activity assay with 4-methylumbeliferyl (4MU)-α-D -glucopyranoside was performed at pH = 4.0. Screening of the iminosugar library on GAA revealed some interesting results. As in Chapters 2 and 3 for GBA2 and GBA1, the iminosugars containing a biphenyl group again show up as potent inhibitors also for GAA. The secondary assay revealed that these iminosugars are able to inhibit GAA at nanomolar levels. Based on the results reported in the previous chapters, it is apparent that the biphenylic iminosugars are not fully selective for GCS and GBA2. The L-ido-congeners of these iminosugars in contrast are selective nanomolar GCS/GBA2 inhibitors, according to the literature, and indeed these compounds, which are also present in the Leiden iminosugar library, do not show up in the here presented screening experiments.
Compounds 22 – 26, that show up in the GAA assays of this chapter, were not identified on screening the same compound library on GBA2 (Chapter 2) and neither on GBA1 (Chapter 3). It should be noted that the screens in the three chapters were done at different final (putative) inhibitor concentrations and it is conceivable that compounds 22 – 26 are also able to inhibit GBA2 at 5 µM. The screens in combination with all the results from the secondary assays have revealed a great amount of valuable information that allows design of more specific GAA inhibitors, taking into consideration also the data from the previous chapters. For instance, in order to increase potency and possibly specificity for GAA, it might be useful to elaborate on the ether motifs in the nitrogen substituents in for instance 23 and 26, and as well on the branched aromatic substituent present in 22.
4.4 Experimental section
Chemicals, materials and methods
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Scheme 2: Synthesis of functionalized 5’/6’-TAMRA (34)
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, 37% over two steps (e) SOCl2 in MeOH, 49%. 6-Azidohex-1-yne (27)
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50.9, 27.8, 25.5, 17.9. HRMS: found 124.0867 [M+H]+, calculated for [C
6H9N3+H]+ 124.0869
Hex-5-yn-1-amine (28)
Triphenylphosphine(1.06 g, 4 mmol, 1 eq.) dissolved in THF (20 mL) was dropwisely added into a solution of 27 (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(31&32) Dimethylaminophenol 29 (6.9 g, 50 mmol, 2 eq.) and trimellitic anhydride 30 (4.8 g, 25 mmol, 1 eq.) 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)-4/5-(hex-4/5-yn-1-yl carbamoyl)benzoate (33)
BOP.PF6 (500 mg, 1.13 mmol, 1.1 eq) and DIPEA (500 µL, 2.87 mmol, 2.5 eq) were added into a mixture containing isomers
31 and 32 (441 mg, 1.03 mmol, 1 eq) and linker 28 (100 mg,
1.03 mmol, 1 eq) dissolved in DMSO (20 mL). The reaction was stirred for 3 days hours at ambient temperature. Both regioisomers were isolated as a mixture 33 (194 mg, 381
µmol, 37% estimated yield over two steps) using HPLC purification. 1H NMR (400
87 Hz, 1H), 7.19 (d, J = 9.4 Hz, 2H), 7.11 (dd, J = 9.5, 2.4 Hz, 2H), 7.03 (d, J = 2.3 Hz, 2H), 3.55 (t, J = 6.9 Hz, 4H), 2.37 – 2.23 (m, 6H), 1.91 – 1.70 (m, 4H), 1.69 (dq, J = 9.9, 7.0 Hz, 4H), 1.33 (s, 2H). 13C NMR (101 MHz, MeOD) δ 166.77, 165.99, 159.37, 157.69, 157.64, 138.12, 136.66, 131.53, 113.49, 68.45, 48.25, 48.07, 47.84, 47.82, 47.43, 47.16, 46.96, 39.29, 28.16, 25.75, 17.38. HRMS: found 510.2386 [M+H]+, calculated for [C31H31O4N3+H]+ 510.2387 N-(6-(dimethylamino)-9-(4/5-(hex-4/5-yn-1-ylcarbamoyl)-2-(methoxycarbonyl) phenyl)-3H-xanthen-3-ylidene)-N-methylmethanaminium (34)
Regioisomeric mixture 33 (50 mg, 98 µmol, 1 eq) was dissolved in 5 mL methanol and cooled to 0°C. To the cooled
mixture 100 µL SOCl2 (164 mg, 1.38 mmol, 14 eq) was added
and the reaction was stirred for 2 hours and warmed to ambient temperature. After overnight reflux the reaction mixture was concentrated under reduced pressure. Regio-isomeric mixture 34 was isolated using HPLC purification (25
mg, 48 µmol, 49% estimated yield over two steps). 1H NMR (400 MHz, MeOD) δ
8.80 (d, J = 1.8 Hz, 1H), 8.29 (dd, J = 8.0, 1.8 Hz, 1H), 7.55 (d, J = 7.9 Hz, 1H), 7.18 (d, J = 9.4 Hz, 2H), 7.09 (dd, J = 9.5, 2.4 Hz, 2H), 7.01 (d, J = 2.3 Hz, 2H), 3.53 (t, J = 6.9 Hz, 4H), 3.44 (s, 6H), 2.37 – 2.23 (m, 6H), 1.91 – 1.70 (m, 4H), 1.69 (dq, J = 9.9, 7.0 Hz, 4H), 1.33 (s, 2H). 13C NMR (101 MHz, MeOD) δ 166.78, 166.01, 159.43, 157.71, 157.62, 138.11, 136.51, 131.53, 113.50, 68.43, 49.86, 48.25, 48.05, 47.83, 47.81, 47.42, 47.17, 46.96, 39.28, 28.17, 25.74, 17.37. HRMS: found 524.2545 [M]+, calculated for [C32H34O4N3]+ 524.2544 (1R,2R,3S,6R)-6-(Hydroxymethyl)-cyclohex-4-ene-1,2,3-triol (4)
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mmol, 57%). 1H-NMR (400 MHz, CD
3OD) δ 5.66 – 5.57 (m, 2H), 4.06 – 4.03 (m, 1H), 3.81 (dd, J = 10.6, 4.1 Hz, 1H), 3.66 - 3.59 (m, 1H), 3.49 - 3.41 (m, 2H), 2.31 - 2.26 (br, 1H); 13C-NMR (100 MHz, CD3OD) δ 130.9, 128.6, 78.8, 73.6, 72.0, 63.5, 47.7 HRMS: found 161.0809 [M+H]+, calculated for [C7H12O4+H]+ 161.0808.
(1R,3R,6R,9S,10S)-9,10-Dihydroxy-3-phenyl-2,4-dioxabicyclo[4.4.0]dec-7-ene (5)
Compound 4 (180 mg, 1.1 mmol, 1.0 eq.) was dissolved in dry DMF (2.0 mL) and dry MeCN (6.0 mL) in an inert atmosphere. Camphorsulfonic acid (52 mg, 0.25 mmol, 0.20 eq.) was added to the solution, followed by PhCH(OMe)2 (253 μL, 1.7 mmol, 1.5 eq.). After 48 h, the reaction was quenched with Et3N (31.0 μL, 0.23 mmol, 0.2 eq.) and concentrated in
vacuo. The reaction mixture was separated out with EtOAc and H2O and the aqueous layer was further extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The product was purified by silica gel column chromatography (30%→70%, EtOAc in pentane) to afford 5 (170 mg, 0.69 mmol, 61%). 1H NMR (400 MHz, CDCl3) δ 7.52 – 7.47 (m, 2H), 7.37 – 7.31 (m, 3H), 5.57 – 5.52 (m, 2H), 5.28 – 5.25 (m, 1H), 4.24 - 4.18 (m, 2H), 3.86 (dd, J = 10.1, 7.5 Hz, 1H), 3.58 – 3.51 (m, 2H), 2.84 (d, J = 8.0 Hz,1H), 2.60 – 2.54 (m, 1H) 13C-NMR (100 MHz, CDCl3) δ 137.8, 130.5, 130.6, 129.2, 128.3, 126.4, 124.1, 102.2, 80.7, 75.4, 73.7, 69.9, 38.5 HRMS: found 249.1122 [M+H]+, calculated for [C14H16O4+H]+ 249.1121. (1R,6R,7R,8R,9S,10R)-10-Hydroxy-7-iodo-3-phenyl-12-trichloromethyl-13-aza-2,4,11 trioxatricyclo [7.4.4.0-8.4.3.0]tridec-12-ene (6)
89 were dried over MgSO4, filtered and concentrated in vacuo. The product was purified by silica gel column chromatography (0%→16%, EtOAc in pentane) to afford 6 (455 mg, 0.88 mmol, 41%). 1H NMR (400 MHz, CDCl3) δ 7.49 – 7.44 (m, 2H), 7.49 – 7.33 (m, 3H), 5.62 (s, 1H), 5.22 (t, J = 7.5 Hz, 1H), 4.85 – 4.83 (m, 1H), 4.72 (s, 1H), 4.21 (dd, J = 11.3, 4.7 Hz, 1H), 4.03 (t, J = 9.8 Hz, 1H), 3.92 (t, J = 10.6 Hz, 1H), 3.83 – 3.79 (m, 1H), 3.08 (s, 1H), 1.20 – 1.13 (m, 1H) 13C NMR (100 MHz, CDCl3) δ 163.7, 137.3, 129.5, 128.5, 126.3, 101.6, 86.7, 77.8, 75.2, 74.9, 72.6, 34.9, 23.7 HRMS: found 519.9150 [M+H]+, calculated for [C16H15Cl3INO4+H]+ 519.9157.
(1S,2R,3S,4R,5R,6R)-5-(Hydroxymethyl)-7-aza-bicyclo-[4.1.0]heptane-2,3,4-triol (7)
A solution of 6 (455 mg, 0.88 mmol, 1.0 eq.) was dissolved in 1,4-dioxane (9.2 mL) and heated to 60°C. Then aqueous HCl (37%, 2.60 mL) was added to the solution. The reaction mixture was stirred at 60°C overnight. The reaction mixture was concentrated in vacuo and then separated out with EtOAc and H2O. The aqueous layer was washed with EtOAc and concentrated in vacuo and co-evaporated with toluene. The crude product of the free amine intermediate was dissolved in MeOH (30 mL) and NaHCO3 (2.9 g, 35 mmol, 40 eq.) was added to the solution. The reaction mixture was stirred at room temperature for 4 days. The reaction mixture filtered and then concentrated in vacuo. The residue was redissolved in H2O and filtered over a pad of Amberlite IR-120 H+ resin, washed with H2O and followed by 1.0 M NH4OH. The filtrate was concentrated in vacuo to afford the aziridine product 7 (97 mg, 0.56 mmol, 63%) as light brown oil. 1H NMR (400 MHz, D2O) δ 4.11 – 4.04 (m, 2H), 3.97 – 3.90 (m, 1H), 3.55 – 3.39 (m, 2H), 2.82 – 2.80 (m, 1H), 2.57 (d, J = 6.4 Hz, 1H), 2.11 – 2.01 (m, 1H). 13C NMR (100 MHz, D2O) δ 73.5, 71.3, 70.1, 61.5, 44.4, 35.7, 31.7 HRMS: found 176.0916 [M+H]+, calculated for [C7H13NO4+H]+ 176.0917.
1S,2R,3S,4R,5R,6R)-2,3,4-trihyrdoxy-5-(hydroxymethyl)-7-(8-azidooctyl)-7-azabicycle[4.1.0]heptane (8)
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1H), 3.66 (dd, J = 8.6, 3.7 Hz, 1H), 3.63 (dd, J = 10.8, 7.1 Hz, 1H), 3.34 – 3.31 (m, 1H), 3.29 (t, J = 6.8 Hz, 2H), 3.05 (t, J = 10.0 Hz, 1H), 2.36 – 2.33 (m, 1H), 2.17 – 2.14 (m, 1H), 1.86 – 1.82 (m, 2H), 1.68 (d, J = 6.5 Hz, 1H), 1.60 – 1.57 (m, 4H), 1.40 – 1.32 (m, 8H) 13C NMR (214 MHz, CD3OD) δ 75.8, 73.4, 72.5, 63.5, 62.3, 52.5, 46.9, 46.0, 41.9, 30.6, 30.5, 30.2, 29.9, 28.4, 27.8 HRMS: found 329.2183 [M+H]+, calculated for [C15H28N4O5+H]+ 329.21833.
N-(6-(dimethylamino)-9-(2-(methoxycarbonyl)-4/5-((4-(1-(8-((1S,2S,3S,4R,5R,6S)-2,3,4-trihydroxy-5-(hydroxymethyl)-7-azabicyclo[4.1.0]heptan-7-yl)octyl)-1H-1,2,3 -triazol-4-yl)butyl)carbamoyl)phenyl)-3H-xanthen-3-ylidene)-N-methylmethan-aminium (ABP 9)
Compound 8 (5 mg, 15 µmol) was
dissolved in DMF (2 mL).
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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 (9, 10 or 11). 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
Recombinant human GAA (Myozyme® from Genzyme) was used during FluoPol-ABPP assays. The optimal probe concentration on FluoPol signal was determined by varying probe concentrations from 1 nM to 250 nM probe at a constant protein concentration (10 µg/mL) and at pH = 5.0. FluoPol-ABPP assays were also performed at different pH values by preparation of different McIlvaine buffers, suppletmented with 0.1% BGG; 0.5 mg/mL Chaps. These pH-experiments were performed at optimal probe concentration (25 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 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 cyclosulphate (9) 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 iminosugar library
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polarization signals were processed as described above. Residual enzyme activities were plotted against the corresponding compound ID.
Analysis of iminosugars as inhibitors of enzymatic activity of GAA and GANAB
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.
GAA: Pure recombinant human enzyme (Myozyme from Genzyme) was used.
Activity was measured with with 3.0 mM 4-methylumbeliferone (4MU)-α-D-glucopyranoside (Sigma) in 150 mM McIlvaine buffer pH 4.0 supplemented with 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.
GANAB: Cellular homogenates of Pompe fibroblasts were used as protein source
for GANAB. Activity was measured with 3.0 mM 4MU-α-D-glucopyranoside in 150 mM McIlvaine pH 7.0 with 0.1% BSA (w/v) for 2 hours. Reactions were stopped as described above for GAA.
4.5 References
(1) Hoefsloot, L. H., Hoogeveen-Westerveld, M., Reuser, A. J. J., and Oostra, B. A. (1990) Characterization of the human lysosomal α-glucosidase gene. Biochem. J.
272, 493–497.
(2) Koshland, D. E. (1953) Stereochemistry and the mechanism of enzymatic reactions.
Biol. Rev. 28, 416–436.
(3) Lee, S. S., He, S., and Withers, S. G. (2001) Identification of the catalytic nucleophile of the Family 31 alpha-glucosidase from Aspergillus niger via trapping of a 5-fluoroglycosyl-enzyme intermediate. Biochem. J. 359, 381–386.
(4) Davies, G. J., Planas, A., and Rovira, C. (2012) Conformational analyses of the reaction coordinate of glycosidases. Acc. Chem. Res. 45, 308–316.
(5) Reuser, A. J. J., Kroos, M. A., Hermans, M. M. P., Bijvoet, A. G. A., Verbeet, M. P., Van Diggelen, O. P., Kleijer, W. J., and van der Ploeg, A. T. (1995) Glycogenosis type II (acid maltase deficiency). Muscle Nerve 18, S61–S69.
(6) van der Ploeg, A. T., and Reuser, A. J. (2008) Pompe’s disease. Lancet 372, 1342– 1353.
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Bourne, Y., Parenti, G., Moracci, M., and Sulzenbacher, G. (2017) Structure of human lysosomal acid α-glucosidase–a guide for the treatment of Pompe disease.
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