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A Specific Activity‐Based Probe to Monitor Family

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A Specific Activity‐Based Probe to Monitor Family DOI:10.1002/cbic.201600561 Full Papers ASpecific Activity-Based Probe to Monitor Family GH59 Galactosylceramidase, the Enzyme Deficient in Krabbe Disease AndrØ R. A. Marques+,[a, f] LianneI.Willems+,[b, g] DanielaHerreraMoro,[a] BogdanI.Florea,[b] SaskiaScheij,[a] RoelofOttenhoff,[a] Cindy P. A. A. van Roomen,[a] Marri Verhoek,[c] JessicaK.Nelson,[a] WouterW.Kallemeijn,[c] AnnaBiela-Banas,[d] Olivier R. Martin,[d] M. BegoÇa Cachón-Gonzµlez,[e] Nee Na Kim,[e] Timothy M. Cox,[e] Rolf G. Boot,[c] Herman S. Overkleeft,*[b] and Johannes M. F. G. Aerts*[a, c] Galactosylceramidase (GALC) is the lysosomal b-galactosidase ring specificity for GALC,equipped with aBODIPY fluorophore responsible for the hydrolysis of galactosylceramide. Inherited at C6 that allows visualization of active enzyme in cellsand tis- deficiency in GALC causes Krabbedisease, adevastating neuro- sues. Detection of residual GALC in patient fibroblasts holds logical disordercharacterizedbyaccumulation of galactosylcer- great promise forlaboratory diagnosis of Krabbedisease.We amide and its deacylated counterpart, the toxic sphingoid base furtherdescribe aprocedure for in situ imaging of active GALC galactosylsphingosine (psychosine). We report the design and in murinebrain by intra-cerebroventricularinfusionofthe ABP. application of afluorescently tagged activity-based probe In conclusion, this GALC-specific ABP should find broad appli- (ABP) for the sensitive and specific labeling of active GALC cations in diagnosis, drug development, and evaluation of molecules from various species. The probe consists of a b-gal- therapyfor Krabbe disease. actopyranose-configured cyclophellitol-epoxide core, confer- Introduction Glycoside hydrolase family 59 (GH59) humangalactosylcerami- the b-anomericconfiguration of the released galactopyrano- dase (GALC, galactocerebrosidase) is an 80 kDa protein respon- side (Scheme 1A). The two carboxylic acid residuesinthe sible for the lysosomalturnover of galactosylceramide andgal- active site that function as anucleophile and ageneral acid/ actosylsphingosine. Newly synthesized GALC contains at least base have been identified as the glutamic acidresidues E258 four N-linked glycosylation sites, which are responsible for lyso- and E182, respectively.[1,3] somal trafficking through the mannose-6-phosphate receptor.[1] Deficiencies in GALC are at the basis of the autosomal reces- After enteringthe lysosomes, the enzyme is cleaved into 30 sive lysosomal storage disorder Krabbe disease, also termed and 50 kDa subunits without effect on enzymatic activity.[2] globoid cell leukodystrophy.More than 70 mutationsinthe Crystalstudies indicate that no dissociation of these subunits gene encoding GALC have been implicated in the develop- occurs.[3] Substrate hydrolysis by GALC occurs through aKosh- ment of this disease.[1] The main pathological consequences land double displacementmechanism with overall retention of are found in the peripheraland central nervous systems. The [a] Dr.A.R.A. Marques,+ D. HerreraMoro, S. Scheij, R. Ottenhoff, [e] Dr.M.B.Cachón-Gonzµlez, N. N.Kim, Prof. Dr.T.M.Cox C. P. A. A.van Roomen, Dr.J.K.Nelson, Prof. Dr.J.M.F.G.Aerts DepartmentofMedicine,University of Cambridge Department of Biochemistry,Academic MedicalCenter Addenbrooke’s Hospital University of Amsterdam Hills Road, CambridgeCB2 2QQ (UK) Meibergdreef 15, 1105 AZ Amsterdam (The Netherlands) [f] Dr.A.R.A.Marques+ [b] Dr.L.I.Willems,+ Dr.B.I.Florea, Prof. Dr.H.S.Overkleeft Present address:Institute of Biochemistry Department of Bio-organic Synthesis Christian-Albrechts-University of Kiel Leiden Institute of Chemistry, Leiden University Otto-Hahn-Platz9,24098 Kiel (Germany) Einsteeinweg55, 2300 RA Leiden (The Netherlands) [g] Dr.L.I.Willems+ E-mail:[email protected] Present address:Department of Chemistry, Simon FraserUniversity [c] M. Verhoek, Dr.W.W.Kallemeijn, Dr.R.G.Boot, Prof.Dr. J. M. F. G. Aerts 8888 University Drive, BurnabyV5A 1S6, BC (Canada) Department of Biochemistry,LeidenInstitute of Chemistry,Leiden University [+] These authorscontributed equally to this work. Einsteinweg 55, 2300 RA Leiden (The Netherlands) Supportinginformation for this article can be found under: E-mail:[email protected] http://dx.doi.org/10.1002/cbic.201600561. [d] Dr.A.Biela-Banas, Prof. Dr.O.R.Martin Institute of Organic and Analytical Chemistry,UniversitØ D’OrlØans Rue de Chartres, B.P. 6759, 45100OrlØans(France) ChemBioChem 2017, 18,402 –412 402 2017 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Full Papers Scheme1.Galactocerebrosidase and structuresofinhibitors and probes 1–7.A)Mechanism of substrate hydrolysisbyGALC. B) Proposed mechanism of GALC binding by compounds 1–4.C)Structures of novel retaining b-galactosidase inhibitor 1 and ABPs 2–4. b-Glucopyranosyl-configured compound 5 targets glu- cocerebrosidase. ABB166 (6)[31] and galactosylsphingosine (psychosine, 7)are competitive GALC inhibitors. mechanism behind this neuropathologyhas not been fully elu- fibroblasts, which is usually 0–5%ofnormallevels.However, cidated.[1,2] Reduced activity of GALC resultsinreduced catabo- the amount of residual activity is neither directly correlated to lism of galactosphingolipids,including galactosylceramide and the clinical symptomsnor to the course of the disease. Carriers galactosylsphingosine (psychosine). Galactosylceramide is the might have as little as 10–20%ofnormalGALC activity with- main lipid component of myelin, the protective sheath around out being affected. The only availabletreatment for Krabbe neuron axons that is essential for correctfunctioning of the disease involves symptomatic treatment and physical therapy. nervoussystem. Accumulation of the toxic metabolite galacto- Clinicaltrials with hematopoietic stem cell transplantation, sylsphingosine eventually leads to apoptosis of myelin-forming which aim to restoreGALC activity in the central nervous cells and consequentdemyelination and neurodegenera- system and therebypreventfurtherdemyelination, hold prom- tion.[4,5] Infantile Krabbe disease is usually diagnosed before ise in slowing the course of juvenile Krabbedisease when di- one year of age and lethal beforethe age of two years. Early agnosed at an early stage.[8,9] symptomsinclude limb stiffness, developmental delay,and Further investigations of GALC and its involvement in severe irritability.When diagnosed in adolescents or adults, Krabbedisease would benefit from the availability of an activi- other symptoms can be observed, such as seizures,feeding dif- ty-based probe (ABP) that specifically targets this enzyme. Nu- ficulties,slowing of mental and motor development, muscle merous fluorescent b-galactosidaseprobeshave been reported weakness, spasticity,deafness, and blindness. The onset and in literature. However, most of these are reversibleand there- severity of symptoms, as well as the course of the disease in fore cannot be used in, for example, gel-based assays. Exam- adult Krabbe patients, is highly variable, even in patients carry- ples include substrates consisting of a b-galactose moiety with ing the same mutation.[6,7] Diagnosis can be confirmed by aluminescentorfluorogenic tagattached to the aglycon posi- measuring residual GALC activity in leukocytes or cultured skin tion that is released after cleavage by the enzyme,[10–16] as well ChemBioChem 2017, 18,402 –412 www.chembiochem.org 403 2017 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim Full Papers as fluorescently tagged competitive inhibitors.[17,18] In addition, Results afew ABPs that enable irreversible mechanism-basedlabeling of retaining b-galactosidases have been reported. These in- Labelingand inhibition of recombinant galactocerebrosi- clude suicide substrates bearing alatent quinone methide pre- dase cursor as the aglycon, to whichafluorescent or fluorogenic tag is attached,[19–22] and 2-fluorogalactoside inhibitors in which First we evaluated the ability of compounds 1–4 to inhibit re- the hydroxy group at C6 is substituted with an azide, which combinant GALC by measuring residual enzyme activity using enablestwo-step labelingbyStaudinger–Bertozzi ligation.[23] the fluorogenicsubstrate 4-methylumbelliferyl b-d-galactopyr- To date, noneofthese probes has been used for the labeling anoside(4-MU b-Gal) after 30 min of pre-incubationwith vary- of human retaining b-galactosidases. ing concentrations of the probes. Plots of residual activity Our work on activity-based retainingglycosidaseprobes against inhibitorconcentration reveal acleardose-dependent used the naturalproduct and retaining b-glucosidaseinhibitor, inhibition of GALC by all probes(Figure 1A). The apparent IC50 cyclophellitol, as starting point.[24] Substituting the epoxidefor values calculated from these curvesare shown in Table1.The an aziridine and grafting areportergroup onto the aziridine nitrogen yielded ABPs that were broadly selectivefor various members within agiven class of retaining glycosidases. Glyco- Table 1. Inhibition of recombinant galactocerebrosidase activity. sidase family selectivity was dictated by the configurationof Compound Apparent ki/Ki Inhibition [%] the cyclophellitol aziridine derivative,anapproachthat was [a] 1 1 IC50 [mm] [mmÀ minÀ ]30min 6h shown valid for GH1-retaining b-glucosidases,GH79-retaining epoxide 1 0.038 12.06 98 (1 mm)100 (1 mm) a-galactosidases and, most recently, for GH29-retaining a-fuco- azido-epoxide 2 70 n.a. 62 (100 mm)96(100 mm) [25–27] sidases. In our first forays into activity-based glycosidase BODIPY-epoxide 3 2.8 0.014 97 (100 mm)100 (100 mm)
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