Macauley, Matthew S.

Stubbs, Keith A.

Vocadlo, David J.

English

Summit Research Repository (Simon Fraser University)

A Selective Inhibitor Gal-PUGNAc of Human Lysosomalb-Hexosaminidases Modulates Levels of the Ganglioside GM2 inNeuroblastoma Cells**Keith A. Stubbs,* Matthew S. Macauley, and David J. Vocadlo*Imbalances in the levels of many glycoconjugates have beenimplicated in human diseases. Many inhibitors of the largesuper family of glycoside hydrolases, which are the enzymesthat break down these glycoconjugates, are known. However,few of these inhibitors have been developed and character-ized as being able to selectively target the enzyme of interestover other functionally related glycoside hydrolases. Suchhighly selective inhibitors are critical tools to gain insightsinto the roles of specific glycoconjugates in disease etiologyand normal cellular function.In humans, there are three enzymes that cleave terminalb-linked N-acetylglucosamine residues from glycoconjugates.The two dimeric isozymes, hexosaminidase A and B (HEXAand HEXB),[1] share high sequence identity and are membersof the family 20 glycoside hydrolases (GH 20). HEXA andHEXB are localized in the lysosome, where they hydrolyzeterminal N-acetylglucosamine and N-acetylgalactosamineresidues of glycosphingolipids such as the ganglioside GM2(Figure 1a), which contains a tetrasaccharide terminating atits non-reducing end with an N-acetylgalactosamine residue.[2]Inheritable mutations impairing either enzyme result in theaccumulation of GM2 in the brain, causing the neurode-generative disorders known as Tay-Sachs and Sandhoffdiseases.[3] Deleterious effects arising from the accumulationof gangliosides are still being uncovered.[4] Interest in thebiological roles played by gangliosides have increased as theirroles in the modulation of cellular signaling have becomeapparent.[5, 6]The other human glycoside hydrolase that cleaves termi-nal b-linked N-acetylglucosamine residues is a family 84enzyme termed O-GlcNAcase (OGA). This enzyme acts onpost-translationally modified nucleocytoplasmic glycopro-teins that bear 2-acetamido-2-deoxy-b-d-glucopyranosideresidues linked to serine or threonine (O-GlcNAc, Fig-ure 1b).[7, 8] The consequences of impairing O-GlcNAcase inmammals are unknown; however, this enzyme has beenimplicated in Alzheimer disease and Type II Diabetes.[8]Although O-GlcNAcase and the lysosomal b-hexosami-nidases act on different substrates and are located in differentcellular compartments, both of these enzymes use a catalyticmechanism involving nucleophilic participation of the 2-acetamido group of the substrate.[9–11] This commonality ofthe catalytic mechanism delayed the development of selectiveO-GlcNAcase inhibitors.[11] However, various inhibitors of O-GlcNAcase[12–15] are proving to be critical tools for evaluatingthe roles played by O-GlcNAc.[16] In contrast, no inhibitor hasyet been described that inhibits b-hexosaminidases in acellular setting to increase levels of GM2. Such a smallmolecule probe would however be a powerful tool fordefining the roles played by gangliosides such as GM2 orfor creating readily titratable models of certain disease states.Figure 1. a) Structure of ganglioside GM2 and b) O-GlcNAc, and c) thecompounds used herein.[*] Dr. K. A. Stubbs,[+] M. S. Macauley, Prof. D. J. VocadloDepartment of Chemistry, Simon Fraser University8888 University Drive, Burnaby, British Columbia V5A 1S6 (Canada)Fax: (+ 1)604-291-3765E-mail: dvocadlo@sfu.ca[+] Current address: Chemistry M313, School of Biomedical, Biomo-lecular and Chemical Sciences, University of Western Australia35 Stirling Highway, Crawley, Western Australia 6009 (Australia)Fax: (+ 618)6488-1005E-mail: kstubbs@cyllene.uwa.edu.au[**] K.A.S. and M.S.M. contributed equally to this work. We thank DonMahuran and Michael Tropak for their generous gift of human HEXA and HEXB, Rosemary Cornell for the HPTLC plates, and Cara-Lynne Schengrund for useful discussions on the use of SK-N-SH cells and HPTLC of gangliosides. This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada (NSERC). D.J.V. is a scholar of the Michael Smith Foundation of Health Research (MSFHR) and a Tier II Canada Research Chair in Chemical Glycobiology. K.A.S. is supported by a Discovery project grant from the Australian Research Council. M.S.M. is a recipient of a senior scholarship from the NSERC and MSFHR.Final version published as: Stubbs, K. A., Macauley, M. S., & Vocadlo, D. J. (2009). A Selective Inhibitor Gal-PUGNAc of Human Lysosomal β-Hexosaminidases Modulates Levels of the Ganglioside GM2 in Neuroblastoma Cells. Angewandte Chemie International Edition, 48(7), 1300–1303. https://doi.org/10.1002/anie.200804583Despite a number of inhibitors having been generated forthese three enzymes (HEXA, HEXB, and O-GlcNAcase), thenumber of synthetic steps and overall yield often limit theiruse in biological studies, as large quantities of these inhibitorsare often required. Furthermore, most of these inhibitorsconcomitantly inhibit both family 20 (GH 20) and family 84(GH 84) enzymes and are thus less useful for studying the roleof one particular enzyme in a biological setting as they mayelicit a complex phenotype. Accordingly, there is a need forpotent inhibitors that are selective for lysosomal b-hexos-aminidases over O-GlcNAcase, and are able to elevate levelsof ganglioside GM2 in cells.One inhibitor of all three enzymes is O-(2-acetamido-2-deoxy-d-glucopyranosylidene)-amino-N-phenylcarbamatePUGNAc.[17] Despite its benefits, one limitation of PUGNAcis its indiscriminate inhibition of both family 20 lysosomal b-hexosaminidases[11, 18] and family 84 O-GlcNAcase.[11, 19] Giventhat HEXA and HEXB can process both gluco- and galacto-configured hexosaminides,[1] while O-GlcNAcase is selectivefor gluco-configured substrates,[19] a galacto-configured C-4epimer of PUGNAc appeared to be a simple route to ensureselectivity by exploiting these differences in substrate specif-icities. We therefore synthesized Galactose-PUGNAc 1 (Gal-PUGNAc), and investigated its activity toward human b-hexosaminidase in enzyme assays and in cultured humanneuronal cells.Starting from galactosamine hydrochloride, Gal-PUGNAc 1 was prepared in eight steps, with an overallyield of nearly 20 % (see Supporting Information), using aroute modified from that previously described for synthesis ofPUGNAc.[12] Gal-PUGNAc 1 was found to be a potent andcompetitive inhibitor of HEXA and HEXB, with an inhib-ition constant (Ki) of 18 1 nm against HEXB (Figure 2) and51 3 nm against HEXA (see Supporting Information). Gal-PUGNAc 1 is therefore comparable to the most potentinhibitors of family 20 b-hexosaminidases that have Ki valuesin the low nanomolar range (see Supporting Informa-tion).[11, 20,21]We evaluated the selectivity of Gal-PUGNAc 1 and foundthat at a concentration 1 mm, Gal-PUGNAc 1 showed lessthan 1 % inhibition of O-GlcNAcase, which is consistent withthe inability of O-GlcNAcase to process galactose-configuredsaccharides.[19] The Ki value of Gal-PUGNAc 1 against O-GlcNAcase must therefore be greater than 10 mm, making itthe most selective inhibitor for lysosomal b-hexosaminidasesover O-GlcNAcase (over 500 000-fold; see Supporting Infor-mation). a-N-Acetylgalactosaminidases are functionallyrelated to b-hexosaminidases in that they also processterminal N-acetylgalactosamine residues, albeit only thosehaving an a-glycosidic linkage. Therefore, to further inves-tigate the selectivity of Gal-PUGNAc 1 we evaluated itspotency towards the G. gallus GH 27 a-N-acetylgalactosami-nidase, which shares 50% identity with the human homologue(NAGA) including completely conserved active site resi-dues[22] and determined a Ki value of 140 10 mm. Gal-PUGNAc 1 is therefore 2800-fold more selective for b-hexosaminidase over the GH 27 a-N-acetylgalactosamini-dase. Gal-PUGNAc 1 also binds over 40 times more tightlythan another galactose-configured inhibitor of lysosomal b-hexosaminidases, Gal-NAG-thiazoline (Figure 1 c), whichwas reported to have a Ki of 820 nm against HEXA.[23] Gal-NAG-thiazoline, however, has not been tested against O-GlcNAcase nor used in cell culture. We therefore preparedthis compound and found that, like Gal-PUGNAc 1, it is alsoa poor inhibitor of O-GlcNAcase.Having demonstrated the selectivity of Gal-PUGNAc 1and Gal-NAG-thiazoline for the lysosomal b-hexosamini-dases in vitro, we evaluated these inhibitors in cultured SK-N-SH cells. These cells are a well-characterized human neuro-blastoma cell line that has appreciable levels of GM2.[24] Cellswere treated with a panel of inhibitors of HEXA, HEXB, andO-GlcNAcase; this panel was comprised of Gal-PUGNAc 1,PUGNAc,[12,17, 25] NAG-thiazoline,[9, 11] Gal-NAG-thiazo-line,[23] and NButGT.[11] After nine days in the presence ofinhibitors, glycosphingolipids were extracted from the cellsand ganglioside levels were assessed by high-performancethin-layer chromatography using resorcinol staining. Levelsof GM2 were significantly elevated (30–40%) in cells treatedwith Gal-PUGNAc 1, PUGNAc, NAG-thiazoline, and Gal-NAG-thiazoline (Figure 3). Notably, NButGT, the O-GlcNA-case specific inhibitor,[11] did not elevate cellular GM2 levels(Figure 3d). This result is consistent with NButGT being apoor inhibitor of HEXB (Ki = 340 mm)[11] and highlights theselectivity of this inhibitor towards O-GlcNAcase.To further investigate the value and specificity of theseinhibitors we examined their effect on cellular O-GlcNAclevels. We treated SK-N-SH cells overnight with the panel ofinhibitors used above and analyzed cell lysates for levels of O-GlcNAc by Western blot. As expected, known O-GlcNAcaseinhibitors used previously in cells, including PUGNAc, NAG-thiazoline, and NButGT, all significantly increased cellular O-GlcNAc levels. In contrast, neither Gal-PUGNAc 1 nor Gal-NAG-thiazoline increased cellular O-GlcNAc levelsFigure 2. Inhibition of human HEXB catalyzed hydrolysis of pNP-GlcNAc by Gal-PUGNAc 1 shows a pattern of competitive inhibition.The concentrations of Gal-PUGNAc 1 (mm) used were 0.15 (~), 0.10(^), 0.050 (&), 0.020 (&), 0.010 (*), and 0.00 units(*). Inset: graph-ical analysis of Ki from plotting KM apparent against the concentrationof Gal-PUGNAc 1. Km,app is the apparent Km value obtained at eachinhibitor concentration.(Figure 4) highlighting their selectivity for HEXA andHEXB.One important observation made here for cell biologistsusing non-selective O-GlcNAcase inhibitors to uncover thebiological roles of O-GlcNAc is that PUGNAc and NAG-thiazoline perturb both O-GlcNAc and ganglioside levels. Afurther cautionary note is that PUGNAc has also beenrecently shown to inhibit an a-N-acetylglucosamindase bac-terial homologue from GH 89 that has high active sitesimilarity with human NAGLU.[26] Therefore, results obtainedusing PUGNAc in cells should be interpreted with these off-target effects in mind, as levels of multiple glycoconjugateswill be perturbed.In summary, Gal-PUGNAc 1 is a readily accessed andselective inhibitor of the human lysosomal b-hexosamini-dases. This selectivity is manifested in the ability of Gal-PUGNAc 1 to increase GM2 ganglioside levels in culturedcells, but to not affect O-GlcNAc levels. We find that Gal-NAG-thiazoline affords similar control over GM2 levels andexpect other b-hexosaminidase inhibitors that permeate intocells, including those that are non-carbohydrate-based,[27] toact similarly. Owing to the greater stability and 40-fold higher[1] D. Mahuran, R. A. Gravel, Adv. Genet. 2001, 44, 145 – 163.[2] E. Klenk, Hoppe Seylers Z. Physiol. 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HPTLC analysis of lipid extracts of cellstreated with 200 mm of each compound for nine days (left panels). Ineach experiment, control cells were grown parallel to treated cells.Magnification of the band representing GM2 is shown on the rightpanels along with densitometric analyses of the intensities of the GM2bands.Figure 4. Gal-PUGNAc 1 and Gal-NAG-thiazoline do not increase levels O-GlcNAc-modified proteins. SK-N-SH cells were treated over-night with 50 mm of each of the compounds and analyzed for levels of O-GlcNAc-modified proteins by Western blot (upper panel). Western blot of cellular b-actin levels demonstrates equal loading for each lane (lower panel).potency of Gal-PUGNAc 1 over Gal-NAG-thiazoline, Gal-PUGNAc 1 offers advantages for studying lysosomal b-hexosaminidase inhibition and ganglioside GM2 in a cellular context without generating a complex chemical phenotype stemming from concomitant inhibition of O-GlcNAcase. 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A Selective Inhibitor Gal‐PUGNAc of Human Lysosomal β‐Hexosaminidases Modulates Levels of the Ganglioside GM2 in Neuroblastoma Cells

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A Selective Inhibitor Gal‐PUGNAc of Human Lysosomal β‐Hexosaminidases Modulates Levels of the Ganglioside GM2 in Neuroblastoma Cells

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