O-GlcNAcase Fragment Discovery with Fluorescence Polarimetry

Article abstract of DOI:10.1021/acschembio.8b00183

The attachment of the sugar N-acetyl-D-glucosamine (GlcNAc) to specific serine and threonine residues on proteins is referred to as protein O-GlcNAcylation. O-GlcNAc transferase (OGT) is the enzyme responsible for carrying out the modification, while O-GlcNAcase (OGA) reverses it. Protein O-GlcNAcylation has been implicated in a wide range of cellular processes including transcription, proteostasis, and stress response. Dysregulation of O-GlcNAc has been linked to diabetes, cancer, and neurodegenerative and cardiovascular disease. OGA has been proposed to be a drug target for the treatment of Alzheimer's and cardiovascular disease given that increased O-GlcNAc levels appear to exert a protective effect. The search for specific, potent, and drug-like OGA inhibitors with bioavailability in the brain is therefore a field of active research, requiring orthogonal high-throughput assay platforms. Here, we describe the synthesis of a novel probe for use in a fluorescence polarization based assay for the discovery of inhibitors of OGA. We show that the probe is suitable for use with both human OGA, as well as the orthologous bacterial counterpart from Clostridium perfringens, CpOGA, and the lysosomal hexosaminidases HexA/B. We structurally characterize CpOGA in complex with a ligand identified from a fragment library screen using this assay. The versatile synthesis procedure could be adapted for making fluorescent probes for the assay of other glycoside hydrolases.

Full text of DOI:10.1021/acschembio.8b00183

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O-GlcNAcase fragment discovery with fluorescence polarimetry
Vladimir S BORODKIN, Karim Rafie, Nithya Selvan, Tonia Aristotelous,
Iva Navratilova, Andrew T. Ferenbach, and Daan M F van Aalten
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00183 • Publication Date (Web): 11 Apr 2018
Downloaded from http://pubs.acs.org on April 12, 2018
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O-GlcNAcase fragment discovery with fluorescence polarimetry
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Vladimir S. Borodkin1,*#, Karim Rafie1#, Nithya Selvan1,3#, Tonia Aristotelous2, Iva Navratilova2,
Andrew T. Ferenbach1 and Daan M. F. van Aalten1*
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1Centre for Gene Regulation and Expression and Division of Biological Chemistry and Drug
Discovery, School of Life Sciences, University of Dundee, Dow Street, Dundee, DD1 5EH, United
Kingdom. 3Present address: Complex Carbohydrate Research Center, University of Georgia, Athens,
GA, United States.
*To whom correspondence should be addressed at vsborodkin@dundee.ac.uk or
dmfvanaalten@dundee.ac.uk
#These authors contributed equally
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Abstract
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The attachment of the sugar DꢀN-acetylglucosamine (GlcNAc) with to specific serine and threonine
residues on proteins is referred to as protein OꢀGlcNAcylation. OꢀGlcNAc transferase (OGT) is the
enzyme responsible for carrying out the modification while OꢀGlcNAcase (OGA) reverses it. Protein
OꢀGlcNAcylation has been implicated in a wide range of cellular processes including transcription,
proteostasis and stress response. Dysregulation of OꢀGlcNAc has been linked to diabetes, cancer,
neurodegenerative and cardiovascular disease. OGA has been proposed to be a drug target for the
treatment of Alzheimer’s and cardiovascular disease given that increased OꢀGlcNAc levels appear to
exert a protective effect. The search for specific, potent and drugꢀlike OGA inhibitors with
bioavailability in the brain is therefore a field of active research, requiring orthogonal highꢀthroughput
assay platforms. Here we describe the synthesis of a novel probe for use in a fluorescence
polarization based assay for the discovery of inhibitors of OGA. We show that the probe is suitable for
use with both human OGA, as well as the orthologous bacterial counterpart from Clostridium
perfringens, CpOGA, and the lysosomal hexosaminidases HexA/B. We structurally characterize
CpOGA in complex with a ligand identified from a fragment library screen using this assay. The
versatile synthesis procedure could be adapted for making fluorescent probes for the assay of other
glycoside hydrolases.
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Introduction
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Protein OꢀGlcNAcylation is the dynamic and reversible modification of specific serine and threonine
side chains on multitude of nucleocytoplasmic and mitochondrial proteins with a single βꢀNꢀ
acetylglucosamine residue1. The attachment of the sugar is catalyzed by the enzyme OꢀGlcNAc
transferase (OGT) and its removal by OꢀGlcNAcase (OGA). Both OGT and OGA are essential
enzymes in mice, with their loss resulting in lethality2-4. Protein OꢀGlcNAcylation is involved in various
cellular processes including transcription5-8, protein stability/degradation9, and stress response10-12; in
most these processes, however, the exact role of OꢀGlcNAcylation is yet to be deciphered.
Dysregulation of OꢀGlcNAc cycling, associated with malfunctions in the production and activity of the
processing enzymes, is a feature of pathological conditions such as diabetes, cancer, Alzheimer’s
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disease and cardiovascular disease1, 9, 13-15
.
The structural characterization of bacterial OGAs and more recently human OGA gave insights into
the binding mode of OꢀGlcNAc proteins16-20. Schimpl et al. showed that glycosylated substrate
peptides bind in a conserved groove in a similar conformation and orientation, adopting a “V”ꢀshaped
conformation with the residue side chains pointing away from the active site, explaining how a single
enzyme can recognize > 1000 different substrates16. Inspection of the structures revealed that the
peptide backbone forms interactions with OGA, in the ꢀ4 through the +3 position surrounding the Oꢀ
GlcNAc site16. Additional hydrophobic interactions are formed by residues in the ꢀ1 and ꢀ2 position
and a surface exposed Tyr189 in CpOGA, which is conserved in the human OGA (Tyr69)16, 17. Studies
revealed that mutation of Tyr69 in human OGA leads to a significant reduction in catalytic activity on
the substrate analogue 4ꢀmethylumbelliferylꢀβꢀDꢀNꢀacetylglucosaminide (4MUꢀGlcNAc) 17.
Structural studies of human OGT21-23 and bacterial orthologues of OGA24, 25 have allowed the
development of inhibitors26-33 to study the function of these enzymes at the molecular and cellular
level. In the case of OGA, potent, cell penetrant inhibitors exist29-33, and have been used for the
identification of OGA as a potential drug target for the treatment of Alzheimer’s disease (AD) and
cardiovascular disease34-36. Hyperphosphorylation of the protein tau resulting in the formation of
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neurofibrillary tangles (NFTs) in AD leading to neuronal loss can be slowed by increasing Oꢀ
GlcNAcylation, which stabilizes tau, preventing its aggregation34. This study was performed using
mice fed with the OGA inhibitor Thiamet G30. A chemically related molecule NButGT29 was shown to
prevent the accumulation of the protein amyloid β, neuroinflammation and memory loss in another
mouse model of AD35. With respect to cardiovascular disease, inhibition of OGA using NAG
thiazolines and NButGT was shown to be protective and attenuate tissue necrosis after
ischaemia/reperfusion injury36, 37. Furthermore, genetic studies have suggested that deletion of oga is
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3,
perinatally lethal although it is not yet clear whether this essentiality is limited to early development
4. While potent and selective, the existing inhibitors of OGA possess a carbohydrate scaffold, which
has poor drugꢀlike properties with respect to Lipinski’s rules. Achieving therapeutic concentrations of
these molecules in vivo requires the administration of large quantities of the drugs. Molecules with
improved stability, pharmacology and penetrability of the bloodꢀbrain barrier are desired and the
search for these is the subject of ongoing research, exemplified by recent studies describing the use
of click chemistry for the rapid generation of potential OGA inhibitor libraries38 and the use of existing
chemical libraries for the discovery of new drugꢀlike inhibitor scaffolds39.
Fluorescence polarization (FP) is one of many techniques used for the analysis of ligandꢀprotein
interactions. The FP technique offers advantages over methods such as surface plasmon resonance
(SPR) and biolayer interferometry (BLI) for the measurement of binding affinities of proteins to
ligands40. It is robust, the quantities of protein/ligands required for an FP assay are comparatively low
and neither proteins nor ligands require to be immobilized to a surface. This makes the FP assay
completely solutionꢀbased and minimizes skewing of binding equilibrium, which could occur by
immobilizing a protein, as this may impose constraints on structural changes that could occur upon
ligand binding40, 41. Also, as a solution based technique, an FP assay is more amenable to high
throughput formats. While the OGA activity assay based on the hydrolysis of the fluorescent substrate
4MUꢀGlcNAc provides a method for the discovery of OGA inhibitors24, 42, the measurement of
inhibition constants for potent inhibitors requires long incubations periods (e.g. ~7 h for CpOGA and
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GlcNAcstatin C)32. An advantage of an FP based OGA assay is direct measurement of binding rather
than loss of activity, reducing assay length. The main limiting factor in the development of an FP
assay is the requirement for a tailorꢀmade fluorescent ligand with high affinity to the protein/receptor
under study, which is what we report here.
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Results and Discussion
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Previously, we established a robust synthetic approach to a family of highly potent and selective
OGA inhibitors, built on 8ꢀalkylamido 2ꢀphenethyl substituted bicyclic sugarꢀimidazole scaffold
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(GlcNAcstatins) . Here we disclose the synthesis of a fluorescently tagged derivative of the potent
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OGA inhibitor GlcNAcstatin B 1 created by the replacement of the 2ꢀphenethyl group of the parent
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molecule with aminopropyl spacer labeled with FITC (hereafter referred to as GlcNAcstatin BF 2 (Fig.
1)).
Figure 1. Structures of GlcNAcstatin B (1) and GlcNAcstatin BF (2)
We also report a novel FP displacement assay using GlcNAcstatin BF as a fluorescent tracer to
measure the binding affinities of existing inhibitors to the bacterial OGA from Clostridium perfringens,
CpOGA, as well as human OGA (hOGA).
We initially devised a concise synthetic scheme to access GlcNAcstatin BF 2 (Scheme 1). We
scheduled the chemoselective installation of a fluorescent tag onto the derivative 3 bearing the
requisite aminopropyl handle that supports the final steps in synthesis of the target compound. To
reach 3 we devised a novel synthetic approach for the construction of the 8ꢀalkylamido 2ꢀsubstituted
sugarꢀimidazole 4 by reversing the order in which the corresponding substituents would be introduced
into the target molecule as compared to the published approach43.
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Scheme 1. Retrosynthetic analysis of GlcNAcstatin BF
Accordingly, we planned to transform the known key 2ꢀiodoꢀmannoseꢀimidazole 6 into GlcNAcꢀ
imidazole derivative 5, which in turn was to be coupled with NꢀBoc propargyl amine in a Sonogashira
reaction giving the intermediate 4. Although this previously unexplored reverse sequence lacks the
flexibility of the previously published approach43, it sufficed for the case where synthesis of the sole
Nꢀacetyl derivative was required.
Previously, the intermediate 6 was prepared by regioselective monoꢀdeiodination of the diiodoꢀ
derivative 6a, which in turn was the product of a challenging bisꢀiodination (8 eq of NIS, 85 °C, 36 h)
of the bicyclic sugarꢀimidazole 6b. The latter process was found to be unsuitable for upscaling,
resulting in formation of varying amounts of the triply iodinated (ortho position in the pꢀmethoxybenzyl
(PMB) group) product, depending on the quality of NIS used. As an alternative route to intermediate
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6, we decided to reinvestigate the cyclization of the linear 4,5ꢀdiiodoimidazole 7, which was previously
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found to be inert in a triflic anhydride mediated process 43 (Scheme 2).
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Scheme 2
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a Reagents and conditions: (a) Tf2O, C2H4Cl2, 65 °C; (b) PPh3, DIAD, THF, 65 °C, 1h, 85%; (c) EtMgBr, THF, 0 °C, 1 h,
89%; (d) TFAꢀH2O 1:9, DCM, 1h, RT, 85%; (e) DPPA, DBU, Tol, 90 °C, 1h, 95%; (f) PPh3, THFꢀH2O, 65 °C, 3 h, then
Ac2O, DIPEA, 16 RT, 85%; (g) HCCCH2NHBoc; Pd(PPh3)4, CuI, Et3N, DMF, 85 °C, 16 h, 53%; (h) H2, Pd/C, MeOH, 2 h,
RT; (i) HCl:TFE 1:50 , 50 °C, 2 h followed by FITC, DIPEA, DMF, RT, 1 h, 65% over two steps.
We assumed that the decreased pKa of the imidazole moiety in 7 would facilitate intramolecular
reactivity under Mitsunobu Nꢀalkylation conditions, giving the bicyclic sugar imidazole 6a directly. The
intermolecular Mitsunobu Nꢀalkylation reaction of imidazoles bearing electronꢀwithdrawing
substituents with different alcohols, including secondary, has several precedents in the literature44, 45
.
Furthermore, Mitsunobu type cyclization of structurally similar sugarꢀderived 1,4ꢀdiols was
consistently used for the synthesis of Cꢀnucleosides including C2ꢀimidazole and benzimidazole
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derivatives46, 47. In our hands, reaction of the linear precursor 7 under standard Mitsunobu conditions
(PPh3, DIAD, 65 °C) resulted in formation of the expected known bicyclic product 6a in excellent yield
(Scheme 2). The reaction was shown to be equally applicable to multiꢀgram scale preparations.
Taken together, the novel intramolecular Mitsunobu Nꢀalkylation of 7 in conjunction with the proven
highly efficient regioselective monoꢀdeiodination of 6a represents a streamlined (and more economic)
access to the key 2ꢀiodoimidazole 6 that supersedes the triflic anhydride mediated cyclization/bisꢀ
iodination sequence of the former approach43.
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We have previously documented the hydrolytic stability of the BDA group in the GlcNAcꢀimidazoles
43. Now we reveal that this phenomenon also works for neutral glycoimidazoles to introduce useful
orthogonality towards acidolysis between PMB and BDA groups. Indeed, using the standard
conditions for the deprotection of the PMB group (aqueous 90% TFA in DCM48) we were able to
selectively remove the 8ꢀO PMB group in 6 without affecting the DBA49 and (less surprisingly) the
TIPS groups to obtain the monohydroxylic compound 8 in 85 % yield (Scheme 2). This
counterintuitive deprotection protocol offers a simplified alternative to the DDQꢀmediated PMB
removal of the original approach43.
Using 8 as the substrate, the stereoselective azide introduction with inversion of configuration
proceeded smoothly according to the established protocol to give gluco-imidazole 9 in a nearly
quantitative yield. Next, a oneꢀpot stepwise Staudinger reduction/acetylation sequence applied to 9
gave the crucial 2ꢀiodoꢀGlcNAcꢀimidazole 5 in high yield. The Sonogashira coupling of derivative 5
with NꢀBoc propargyl amine resulted in a fair yield (53%) of the desired advanced intermediate 4.
Overall, we have successfully implemented a novel synthetic approach for the construction of 8ꢀ
alkylamidoꢀ2 substituted sugar–imidazoles. This approach produces comparable efficiency to the
original one on all the separate steps but Sonogashira coupling, where somewhat diminished yield
was recorded.
The concluding transformation of 4 into the targeted fluorescent tracer 2 was initiated with
hydrogenation of the triple bond to give the Cꢀ2 aminopropyl equipped derivative 10. The pivotal oneꢀ
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pot global removal of the protecting groups in 10 was efficiently achieved with 12 M HCl in
trifluoroethanol (1:50) at 55 °C for 2 h to give the fully deprotected compound 350, 51 which was
(without purification) treated with a slight excess of FITC in DMF to furnish the fluorescently tagged
product 2 (Scheme 2). The reaction mixture was directly purified by reverse phase flash
chromatography on a C18 column and the product was reꢀpurified to homogeneity by HPLC to afford
the desired GlcNAcstatin BF in 65% yield over the threeꢀstep sequence.
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We next wanted to assess the use of GlcNAcstatin BF with CpOGA and hOGA in an FP assay.
GlcNAcstatin BF bound to CpOGA with a Kd of 0.5 ± 0.2 nM (Fig. 2a) and to hOGA with a Kd of 143 ±
29 nM (Fig. 2b). We then used GlcNAcstatin BF in displacement assays to measure the Kd of the
inhibitors GlcNAcstatin B and GlcNAcstatin G. Consistent with previous reports 32, 33, GlcNAcstatin B
was a better binder of CpOGA with a Ki of 6.6 nM versus 16.3 nM for GlcNAcstatin G (Fig. 2c). For
hOGA, however, the Ki obtained for GlcNAcstatin B was 23.2 nM and for GlcNAcstatin G, it was 21.4
nM (Fig. 2d). The absolute Ki values obtained for these inhibitors are not consistent with previously
reported Ki values obtained using the 4MUꢀNAG based activity assay32, 33. In addition to the variability
expected from using different methods, this could be due to the differences in the assay buffer and
pH used or the different Nꢀterminal truncation of the construct used in this study. Dorfmueller et al.,
used the McIlvaine buffer system at pH 5.7/7.3 32, 33, while here we use Tris buffered saline (TBS) at
pH 7.5. The Ki values obtained with the FP assay for the binding of CpOGA to GlcNAcstatin G and
another potent OGA inhibitor Thiamet G30 are consistent with those obtained using surface plasmon
resonance (SPR) performed using the same assay buffer (Supplementary Fig. S1); the binding
affinity of hOGA for Thiamet G using the FP assay is shown in Supplementary Fig. S2. These proofꢀ
ofꢀconcept experiments establish the fluorescence polarization displacement assay using
GlcNAcstatin BF as a convenient method for evaluating affinity of OGA inhibitors.
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Figure 2. Binding affinity of GlcNAcstatin BF and two inhibitors of the GlcNAcstatin family to CpOGA and hOGA.
FP assay showing the binding of GlcNAcstatin BF to a) CpOGA and b) hOGA. Binding was measured by incubating for 10
min (CpOGA) and 180 min (hOGA) at a fixed concentration of labeled probe ([GBFCpOGA] = 0.5 nM, [GBFhOGA] = 50 nM)
with varying concentrations of enzyme. Data points were fitted to a oneꢀsite specificꢀbinding equation using Prism
(GraphPad). Experiments were performed in triplicate and error bars represent standard error of the mean. Doseꢀ
response curves from the fluorescence polarization assay showing the displacement from c) CpOGA or d) hOGA of a
fixed concentration of fluorescent probe by increasing concentrations of GlcNAcstatin B or GlcNAcstatin G. Highest
amount of probe bound to enzymes in the absence of inhibitors was set as 100%. Data points were fitted to a fourꢀ
parameter equation for doseꢀdependent inhibition using Prism (GraphPad). Experiments were performed in triplicate and
error bars represent standard error of the mean. A summary including Hill Slopes can be found in Supplementary Material
Table 1.
To expand the applicability of the suggested assay system for the highꢀthroughput screening of
custom compound libraries, we decided to evaluate the Maybridge Ro3 1000 fragment library towards
CpOGA as a target. This library consists of fragments that adhere to the ‘rule of three’ (Ro3)
(molecular weight ≤ 300 Da, no more than 3 hydrogen bond donors/acceptors, cLogP ≤ 3). The
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screen was performed under experimental conditions matching those used in the displacement of
GBF by Thiamet G and GlcNAcstatin G (Supplementary material Fig. S3).
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The quality of both screens was estimated by calculating Z′ value across the controls on a given
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throughput assay system. In our hands, we consistently saw Z′ values for each plate between 0.73
– 0.84 with respect to the standard readings, with a Z′ value of 0.66 for readings across all four
plates. We chose to identify compounds as hits if they reduced the maximum polarization by ≥ 40%,
resulting in a list of 15 initial hits. Subsequently, the initial hits were advanced into the second round
of testing, in which we determined the Ki values by measuring the displacement of GlcNAcstatin BF in
a doseꢀdependent manner. Out of the 15 initial hits (F1ꢀF15, Supplementary Material Fig. 3,
Supplementary Material Table 2), 8 were confirmed as binders of CpOGA with apparent Ki values
between 9 – 150 ꢁM.
Binding of a fragment hit to Tyr189 and Asp401 blocks GlcNAcstatin BF and substrate binding
To further validate the fragments identified as binders in the HTS screen, we performed
macromolecular xꢀray crystallography to identify the binding mode of a subset of these hits. We
managed to solve the structure of CpOGA in complex with 5ꢀ(trifluoromethyl)ꢀ2,3ꢀdihydroꢀ1Hꢀ1,4ꢀ
diazepine (Fragment F8 hereafter referred to as 5TFD, apparent Ki = 146 ꢁM, Fig. 3A). We collected
synchrotron diffraction data to 2.6 Å (Supplementary material Table 3) allowing structure solution by
molecular replacement and subsequent refinement (final Rwork/Rfree = 0.17/0.22). There was
continuous |Fo|-|Fc| electron density for the fragment (Fig. 3B). 5TFD binds proximal to the active site
pocket, forming hydrophobic stacking interactions with Tyr189 (Fig 3B). Additionally, the secondary
amine of 5TFD forms a hydrogen bond with the backbone carbonyl oxygen of Asp401 (Fig. 3B). Tyr189
has been previously identified to be important for substrate binding, forming hydrophobic interactions
with the substrate peptide backbones16. When compared to a structure of CpOGA in complex with
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52
GlcNAcstatin F (Fig. 3 C, PDBID: 2XPK ), a nM inhibitor of CpOGA designed to mimic the reaction
intermediate, 5TFD forms different interactions. However, taking into account the longer linker and
presence of the fluorescein group present in GlcNAcstatin BF it is possible for 5TFD to form a steric
barrier for proper GBF binding. Thus, the 5TFD fragment bound to Tyr189 and Asp401, is able to block
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[5TFD] / M
Figure 3. Binding affinity of 5FTD and binding mode of 5FTD and GlcNAcstatin C to CpOGA. a) Doseꢀresponse
curves from the fluorescence polarization assay showing the displacement from CpOGA of a fixed concentration of
fluorescent probe by increasing concentrations of 5FTD. Highest amount of probe bound to enzymes in the absence of
inhibitors was set as 100% and a fictive concentration of 10 M set as 0%. Data points were fitted to a fourꢀparameter
equation for doseꢀdependent inhibition using Prism (GraphPad). Experiments were performed in triplicate and error bars
represent standard error of the mean. A summary including Hill Slopes can be found in Supplementary Material Table 2.
Structure of CpOGA in complex with b) 5FTD and c) GlcNAcstatin F (PDBID 2XPK 52). The protein is shown as grey
cartoon with active site residues shown as sticks. Ligands are shown as green sticks. Hydrogen bonds between ligands
and active site residues are shown as dashed black lines. The unbiased |Fo|-|Fc| electron density map for 5FTD is shown
as blue mesh contoured at 2 σ.
GlcNAcstatin BF may not only be useful to measure the binding of ligands to OGA, but also to
enzymes of related glycoside hydrolase families such as GH20, to which human HexA/B belong. This
is because GlcNAcstatin B itself, while potent, is not a selective inhibitor of OGA32 and also targets
HexA/HexB. We were able to determine an apparent Kd of ~ 4 ꢁM (Supplementary Material Figure 4)
for GlcNAcstatin BF to HexA/B isolated from bovine kidneys. HexA/HexB have recently been
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explored as targets for enzyme enhancement therapy to treat the lysosomal storage diseases Tayꢀ
Sachs and Sandhoff disease53 and an FP based assay offers an alternative to activity assays for the
screening of fragments/compounds that bind to these proteins.
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Here we have reported the design and synthesis of the novel fluorescent probe GlcNAcstatin BF to
support miniaturized OGA highꢀthroughput assays. To synthesize the target compound we
established a novel synthetic approach for the construction of the 8ꢀN alkylamide 2ꢀsubstiuted sugar–
imidazoles by reversing the order of the introduction of the respective substituents into the key
intermediate as compared to the original approach. As part of the study, we also developed a novel
method for the synthesis of bicyclic sugarꢀimidazoles via intramolecular Mitsunobu Nꢀalkylation of 4,5ꢀ
diiodoimidazoles to supersede the challenging sequence of the former approach. We successfully
applied a simplified counterintuitive acid hydrolysis procedure for the removal of the PMB protection
in the presence of cyclic BDA, the product of the unique substrateꢀinduced orthogonality between
PMB and DBA groups. Notably, we established a fast and high yielding procedure for the global
deprotection of the penultimate synthetic intermediate including removal of the notoriously stable BDA
group that constitutes an expedient alternative to the TFA based method previously used for the
GlcNAcstatin synthesis. We have shown that GlcNAcstatin BF is suitable for use in fluorescence
polarization assays to measure the binding affinity of OGA inhibitors and identify novel binders from a
highꢀthroughput screen of a 1000ꢀmember fragment library. Finally, we solved the structure of
CpOGA in complex with a binder identified in the screen and were able to show that it binds to
Tyr189, an important residue for substrate binding.
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Materials and Methods
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Crystallography and structure solution
CpOGA was concentrated to 40 mg mLꢀ1 in 25 mM TrisꢀHCl pH 8.0, 20 mM NaCl, 0.5 mM TCEP.
Sittingꢀdrop vapor diffusion crystallization experiments were performed by mixing drops in a 1:1 ratio
of CpOGA and 0.175 M CdSO4, 0.1 M sodium acetate pH 7.5 and needle shaped crystals appeared
after 3ꢀ4 days. A 5FTD fragment complex was achieved by transferring crystals into a drop containing
10 mM 5FTD in 0.175 M CdSO4, 0.1 M sodium acetate pH 7.5, 1% DMSO for 4 h prior to
cryoprotection with 20% glycerol in mother liquor saturated with 5TFD. Diffraction data was collected
at the European Synchrotron Radiation Facility (ESRF) on beamline ID30Aꢀ3, were processed with
XDS54 and scaled to 2.6 Å using aimless55. 5% of total reflections were set aside as an Rfree test set.
Crystals belonged to space group P61 with one molecule per asymmetric unit, a solvent content of
72% and a Matthews coefficient of 4.5. The structures were solved with MOLREP56, using chain A of
PDB 2YDS16 as a search model. The structure was fully refined using iterative cycles of Refmac557
and COOT58. Ligand topology was generated using PRODRG59. Xꢀray diffraction data collection and
structure refinement statistics can be found in the supplementary material (Supplementary Material
Table 3).
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Fluorescence Polarization
Experiments were performed in PerkinElmer, black, 384ꢀwell plates and millipolarization units
measured using a Pherastar FS plate reader (BMG LABTECH) at excitation and emission
wavelengths of 485 nm and 520 nm, respectively. For determination of the equilibrium dissociation
constant (Kd) of CpOGA and hOGA for GlcNAcstatin BF, 0.5 nM/50 nM of the probe was incubated
with a range of concentrations of protein in 25 ꢁL/ 30ꢁL total reaction volume containing 1 x TBS (25
mM Tris, 150 mM NaCl, pH 7.5) buffer and a final concentration of 1ꢀ2% DMSO. Reactions were
allowed to stand at room temperature for 10 min for CpOGA and 3 h for hOGA, after which
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polarization was measured (equilibrium was reached within these time points). Readings were
corrected for background emissions from reactions without enzyme and the Kd was determined by
fitting a nonꢀlinear regression curve with Prism (GraphPad). To avoid receptor depletion, reaction
mixtures for competition binding experiments contained 1 nM fluorescent probe for CpOGA and 50
nM for hOGA, 7 nM of CpOGA/250 nM hOGA (receptor) and a range of concentrations of inhibitors in
the aforementioned reaction conditions. The largest amount of fluorescent probe bound to the
receptors in the absence of competing ligands was set as 100%. IC50 values were determined by
fitting doseꢀresponse curves with Prism (GraphPad) and converted to Kd as outlined elsewhere60. All
experiments were performed in triplicate.
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High-throughput screen of the Maybridge Ro3 1000 fragment library
The Maybridge Ro3 1000 fragment library (Maybridge) screen was performed in black, 384ꢀwell
plates (PerkinElemer). Displacement of GlcNAcstatin BF was measured by adding 25 ꢁL of 7 nM
CpOGA and 1 nM GlcNAcstatin BF in assay buffer (0.1 M TrisꢀHCl pH 7.4, 150 mM NaCl, 1% DMSO)
to assay plates containing 50 nL of a 0.1 M fragment solutions in DMSO, resulting in a final assay
concentration of 200 ꢁM. The plates were allowed to stand for 10 minutes in the dark before reading
polarization on a Pherastar FS plate reader (BMG Labtech) at excitation and emission wavelengths of
485 nm and 520 nm, respectively. Readings were corrected for background polarization from
reactions containing only 1 nM GlcNAcstatin BF in assay buffer and normalised to readings
containing 1% DMSO. Fragments displacing GlcNAcstatin BF by ≥ 40% were classified as hits and
were advanced for Ki value determination. Competition binding experiments were conducted under
the same assay conditions in 0.1 M TrisꢀHCl pH 7.4, 150 mM NaCl, 2% DMSO and varying
concentrations of fragments. Ki values were calculated as described above.
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Accession codes
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The atomic coordinates and structure factors have been deposited in the Protein Data Bank61 under
the accession code 5OXD.
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Supporting information
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The Supporting Information, including synthetic procedures and spectral data for all new compounds,
methods, figures and tables as well as the compound characterization checklist and the validation
report of the deposited structure are available free of charge via the Internet.
Acknowledgements
We thank the European Synchrotron Radiation Facility (ESRF) for beam time on ID30Aꢀ3 and
assistance. This work was funded by a Wellcome Trust Senior Research Fellowship (WT087590MA)
to DMFvA. We would like to hank Dr. M. Schimpl for purifying hOGA.
Author Contributions
The study was conceived by VSB, NS and DMFvA. Synthesis was performed by VSB; protein
purification, protein biotinylation and FP assays were performed by NS; KR performed HTS, FP and
hitꢀvalidation assays and structural biology; cloning was performed by ATF and SPR was performed
by TA and IHN. Data were analysed by all authors. The manuscript was written by VSB, NS, KR and
DMFvA.
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O
S
OH
N
H
N
HO1
N
H
O
OGA inhibitors
O
N
HO
HO
NHAc
OH
2
3
4
5
6
7
High affinity fluorescent tracer
Hightroughput OGA
Fluorescent Polarization Assay
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