Fluorescence polarisation activity-based protein profiling for the identification of deoxynojirimycin-type inhibitors selective for lysosomal retaining alpha- and beta-glucosidases

Lysosomal exoglycosidases are responsible for processing endocytosed glycans from the non-reducing end to produce the corresponding monosaccharides. Genetic mutations in a particular lysosomal glycosidase may result in accumulation of its particular substrate, which may cause diverse lysosomal storage disorders. The identification of effective therapeutic modalities to treat these diseases is a major yet poorly realised objective in biomedicine. One common strategy comprises the identification of effective and selective competitive inhibitors that may serve to stabilize the proper folding of the mutated enzyme, either during maturation and trafficking to, or residence in, endo-lysosomal compartments. The discovery of such inhibitors is greatly aided by effective screening assays, the development of which is the focus of the here-presented work. We developed and applied fluorescent activity-based probes reporting on either human GH30 lysosomal glucosylceramidase (GBA1, a retaining β-glucosidase) or GH31 lysosomal retaining α-glucosidase (GAA). FluoPol-ABPP screening of our in-house 358-member iminosugar library yielded compound classes selective for either of these enzymes. In particular, we identified a class of N-alkyldeoxynojirimycins that inhibit GAA, but not GBA1, and that may form the starting point for the development of pharmacological chaperone therapeutics for the lysosomal glycogen storage disease that results from genetic deficiency in GAA: Pompe disease.


Crystallographic studies of rhGBA1
A co-crystal structure of rhGBA1 in complex with ABP-IV (MDW933) was obtained at 1.86 Å resolution to reveal a single molecule of ABP-IV bound covalently to the catalytic nucleophile (Glu340) of both rhGBA1 chains in the crystallographic dimer.Specifically, the cyclophellitol moiety reacts with Glu340 through its epoxide warhead, to form a covalent trans-diaxial ring opened cyclitol in the 4 C1 chair conformation (Fig. S2).Whilst only the reacted cyclitol and triazole linker of the ABP molecule bound in chain B could be modelled (likely due to disorder of the alkyl linker and/or probe decomposition), sufficient electron density was observed to model the full, intact probe bound in chain A. The C6-triazole linker and subsequent 4-carbon alkyl chain were modelled through a broad active site cleft, formed by Tyr244, Pro245, Phe246, Tyr313 and Asn396, which extends towards the dimer interface where the BODIPY tag binds (Fig. S3).The BODIPY binds in a hydrophobic cavity at the dimer interface formed by residues Leu241, Leu314, Phe316, Phe347 and Trp348 of rhGBA1 chain A and residues Leu241, Leu314, Phe316 and Leu317 of rhGBA1 chain B, consistent with the binding of a Cy5tagged ABP we reported previously 15 and with a recently reported serendipitous co-complex of ABP-IV and N-acyl cyclophellitol aziridine (KY358). 16Electron density was also observed for the BODIPY tag and the triazole-alkyl linker of a decomposed probe bound in a distant site on the surface of the TIM-barrel domain of rhGBA1 (S4).

Production and crystallization of rhGBA1
Recombinant human GBA1 (rhGBA1) was produced in an insect-baculovirus expression system and purified according to previously published procedures. 17rhGBA1 was subsequently crystallised in a 48-well MRC sitting-drop vapour-diffusion format using previously reported conditions containing 0.2 µL GBA1 (10 mg.mL −1 ) + 0.

Data collection, structure solution and refinement
Data were collected at the i03 beamline of the Diamond Light Source (DLS) UK and processed using XIA2 18 and AIMLESS 19,20 data reduction pipelines through the CCP4i2 suite. 21The structure was solved by molecular replacement using MOLREP with the previously deposited unliganded structure (PDB 6TJK) 17 as homologous search model.
Structure refinement was performed using REFMAC 22 followed by several rounds of manual model building with COOT. 23,24Idealized coordinate sets and refinement dictionaries for the ligand were generated using ACEDRG 25,26 in the CCP4 suite.Conformation of all sugars were validated using Privateer 27 and the structure was validated using MolProbity 28 and the wwPDB Validation service (validate-rcsb-1.wwpdb.org/)prior to deposition.All crystal structure figures were generated using CCP4 mg. 29Data collection and refinement statistics are summarised in Table S3.

Crystallographic studies of rhGAA in complex with ABP-II
The structure of rhGAA (Myozyme) soaked with ABP-II has been obtained at 1.9 Å resolution.As can be seen in Fig. S5, during the time-laps of rhGAA crystals bathing in crystallization solution supplemented with ABP-II, the rhGAA nucleophile Asp518 operated a nucleophilic attack on the epi-cyclophellitol aziridine warhead, leading to a covalent and irreversible enzyme-inhibitor complex where the cyclitol moiety of ABP-II adopts a 1 S3 chair conformation.The cyclitol hydroxyl groups establish the same hydrogen-bonding interactions as described for the rhGAA-N-PNT-DNM 15 complex in the main text.The nitrogen from the reacted aziridine establishes two water mediated contacts with Asp616 and Asp282, respectively.The alkyl chain is lined with Met519, Trp376, Trp481 and Phe525, and the triazole ring stacks against Phe525.Finally the subsequent alkyl chain blots against Phe525 and Val480 and the terminal nitrogen atom, last atom to be seen in the electron density map, establishes a hydrogen-bond interaction with the main-chain carbonyl of Val480.The remaining part of the probe, notably the TAMRA group, could not be observed in the electron density, most likely due to structural disorder.Globally, it appears that Phe525 is a major player in ABP-II recognition, where the ensemble of alkyl chains and the triazole adduct wrap around the side chain of this amino-acid.In summary it can be noticed that the ABP-II probe accommodates perfectly within the rhGAA substrate-binding groove, making it a suitable activity-based probe for GAA.

rhGAA sample preparation, crystallization and crystallographic procedures
Samples of recombinant human GAA (rhGAA) were kindly provided by the teams of Giancarlo Parenti and Marco Moracci, University of Naples.Shortly, these were residual amounts of the infusions of Myozyme® (Sanofi Genzyme, Cambridge, MA) administrated for the treatment of Pompe patients at the Department of Translational Medical Sciences, Federico II University, Naples, Italy.Sample conditioning and crystallization were performed as described

S44
previously. 30Crystal soaking was achieved by transferring crystals of rhGAA to small drops composed of crystallization liquor and either compound 15 or ABP-II at a final concentration of 10 mM, followed by incubation for ~3 hours.Crystals were then cryo-protected with reservoir solution supplemented with 30% (v/v) glycerol prior flash cooling in liquid N2.X-ray diffraction data have been acquired at beam line Proxima2, Synchrotron Soleil, Gif-sur-Yvette, France, and processed with XDS 31 and the CCP4 software suite. 21Structures were obtained by difference Fourier synthesis with REFMAC 22 using the native structure of rhGAA (PDB entry 5NN3) as starting model.Ligand coordinates were generated with jLigand. 32Models were refined with subsequent rounds of Refmac 22 and Coot 24 respectively.Indices for the Rfree cross-validation data sets were taken over from PDB entry 5NN4 and extended to 1.75 Å resolution for the complex with compound 15.Model quality was assessed with internal modules of Coot 24 and with the Molprobity server. 28

Figure S3 .FluorescenceS4Figure S4 .
Figure S3.Ribbon diagram of the rhGBA1 dimer with surface depiction of the BODIPY tag of ABP IV bound at the dimer interface (red surface).

Figure S8 .
Figure S8.Inhibition curves using 4-MU alpha glucoside as fluorogenic substrate.Two replicates are shown for compounds 6 and 13-15 in rhGAA (A) and GANAB (B) where residual enzyme activity (%) versus the logarithm of different concentrations of inhibitors is plotted (SeeTable 1 for IC50 values).

Fluorescence
Polarisation Activity-Based Protein Profiling for the Identification of Deoxynojirimycintype Inhibitors selective for Lysosomal Retaining Alpha-and Beta-Glucosidases van der Gracht et al.S6

Table S1 .
Chemical structures of the 358 compounds of the Leiden iminosugar library which has been screened in the GBA1 and GAA FluoPol ABPP assays.Fluorescence Polarisation Activity-Based Protein Profiling for the Identification of Deoxynojirimycintype Inhibitors selective for Lysosomal Retaining Alpha-and Beta-Glucosidases van der Gracht et al.Fluorescence Polarisation Activity-Based Protein Profiling for the Identification of Deoxynojirimycintype Inhibitors selective for Lysosomal Retaining Alpha-and Beta-Glucosidases van der Gracht et al.Fluorescence Polarisation Activity-Based Protein Profiling for the Identification of Deoxynojirimycintype Inhibitors selective for Lysosomal Retaining Alpha-and Beta-Glucosidases van der Gracht et al.Fluorescence Polarisation Activity-Based Protein Profiling for the Identification of Deoxynojirimycintype Inhibitors selective for Lysosomal Retaining Alpha-and Beta-Glucosidases van der Gracht et al.Fluorescence Polarisation Activity-Based Protein Profiling for the Identification of Deoxynojirimycintype Inhibitors selective for Lysosomal Retaining Alpha-and Beta-Glucosidases van der Gracht et al.Fluorescence Polarisation Activity-Based Protein Profiling for the Identification of Deoxynojirimycintype Inhibitors selective for Lysosomal Retaining Alpha-and Beta-Glucosidases van der Gracht et al.Fluorescence Polarisation Activity-Based Protein Profiling for the Identification of Deoxynojirimycintype Inhibitors selective for Lysosomal Retaining Alpha-and Beta-Glucosidases van der Gracht et al.Fluorescence Polarisation Activity-Based Protein Profiling for the Identification of Deoxynojirimycintype Inhibitors selective for Lysosomal Retaining Alpha-and Beta-Glucosidases van der Gracht et al.
Fluorescence Polarisation Activity-Based Protein Profiling for the Identification of Deoxynojirimycintype Inhibitors selective for Lysosomal Retaining Alpha-and Beta-Glucosidases van der Gracht et al.Fluorescence Polarisation Activity-Based Protein Profiling for the Identification of Deoxynojirimycintype Inhibitors selective for Lysosomal Retaining Alpha-and Beta-Glucosidases van der Gracht et al.Fluorescence Polarisation Activity-Based Protein Profiling for the Identification of Deoxynojirimycintype Inhibitors selective for Lysosomal Retaining Alpha-and Beta-Glucosidases van der Gracht et al.Fluorescence Polarisation Activity-Based Protein Profiling for the Identification of Deoxynojirimycintype Inhibitors selective for Lysosomal Retaining Alpha-and Beta-Glucosidases van der Gracht et al.Fluorescence Polarisation Activity-Based Protein Profiling for the Identification of Deoxynojirimycintype Inhibitors selective for Lysosomal Retaining Alpha-and Beta-Glucosidases van der Gracht et al.Fluorescence Polarisation Activity-Based Protein Profiling for the Identification of Deoxynojirimycintype Inhibitors selective for Lysosomal Retaining Alpha-and Beta-Glucosidases van der Gracht et al.Fluorescence Polarisation Activity-Based Protein Profiling for the Identification of Deoxynojirimycintype Inhibitors selective for Lysosomal Retaining Alpha-and Beta-Glucosidases van der Gracht et al. 6 Table S2.Iminosugars identified in the rhGAA FluoPol-ABPP assay.Residual FP-signal (%).IC50 values for in vitro inhibition of lysosomal rhGAA (Myozyme) and ER--glucosidase II (GANAB) using 4-MU alpha glucoside substrate.

Table S3 .
Crystallographic data collection and refinement statistics.Based Protein Profiling for the Identification of Deoxynojirimycintype Inhibitors selective for Lysosomal Retaining Alpha-and Beta-Glucosidases van der Gracht et al.
Fluorescence Polarisation Activity-Based Protein Profiling for the Identification of Deoxynojirimycintype Inhibitors selective for Lysosomal Retaining Alpha-and Beta-Glucosidases van der Gracht et al.
Fluorescence Polarisation Activity-Based Protein Profiling for the Identification of Deoxynojirimycintype Inhibitors selective for Lysosomal Retaining Alpha-and Beta-Glucosidases van der Gracht et al.
Figures were generated with Pymol (The PyMOL Molecular Graphics System, Version 2.3.5, Schrödinger, LLC.).Polarisation Activity-Based Protein Profiling for the Identification of Deoxynojirimycintype Inhibitors selective for Lysosomal Retaining Alpha-and Beta-Glucosidases van der Gracht et al.H-NMR and 13 C-NMR spectra of 5'-TAMRA ABP I in MeOD Fluorescence Polarisation Activity-Based Protein Profiling for the Identification of Deoxynojirimycintype Inhibitors selective for Lysosomal Retaining Alpha-and Beta-Glucosidases van der Gracht et al.S47 1 H-NMR and 13 C-NMR spectra of 5'-and 6'-TAMRA ABP II in MeOD Fluorescence Polarisation Activity-Based Protein Profiling for the Identification of Deoxynojirimycintype Inhibitors selective for Lysosomal Retaining Alpha-and Beta-Glucosidases van der Gracht et al.