Discovery of a Novel and Brain-Penetrant O-GlcNAcase Inhibitor via Virtual Screening, Structure-Based Analysis, and Rational Lead Optimization

Article abstract of DOI:10.1021/acs.jmedchem.0c01712

O-GlcNAcase (OGA) has received increasing attention as an attractive therapeutic target for tau-mediated neurodegenerative disorders; however, its role in these pathologies remains unclear. Therefore, potent chemical tools with favorable pharmacokinetic profiles are desirable to characterize this enzyme. Herein, we report the discovery of a potent and novel OGA inhibitor, compound 5i, comprising an aminopyrimidine scaffold, identified by virtual screening based on multiple methodologies combining structure-based and ligand-based approaches, followed by sequential optimization with a focus on ligand lipophilicity efficiency. This compound was observed to increase the level of O-GlcNAcylated protein in cells and display suitable pharmacokinetic properties and brain permeability. Crystallographic analysis revealed that the chemical series bind to OGA via characteristic hydrophobic interactions, which resulted in a high affinity for OGA with moderate lipophilicity. Compound 5i could serve as a useful chemical probe to help establish a proof-of-concept of OGA inhibition as a therapeutic target for the treatment of tauopathies.

Full text of DOI:10.1021/acs.jmedchem.0c01712

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Article
Discovery of a Novel and Brain-Penetrant O‑GlcNAcase Inhibitor via
Virtual Screening, Structure-Based Analysis, and Rational Lead
Optimization
Michiko Tawada,* Makoto Fushimi, Kei Masuda, Huikai Sun, Noriko Uchiyama, Yohei Kosugi,
Weston Lane, Richard Tjhen, Satoshi Endo, and Tatsuki Koike
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ABSTRACT: O-GlcNAcase (OGA) has received increasing attention as an attractive therapeutic target for tau-mediated
neurodegenerative disorders; however, its role in these pathologies remains unclear. Therefore, potent chemical tools with favorable
pharmacokinetic profiles are desirable to characterize this enzyme. Herein, we report the discovery of a potent and novel OGA
inhibitor, compound 5i, comprising an aminopyrimidine scaffold, identified by virtual screening based on multiple methodologies
combining structure-based and ligand-based approaches, followed by sequential optimization with a focus on ligand lipophilicity
efficiency. This compound was observed to increase the level of O-GlcNAcylated protein in cells and display suitable
pharmacokinetic properties and brain permeability. Crystallographic analysis revealed that the chemical series bind to OGA via
characteristic hydrophobic interactions, which resulted in a high affinity for OGA with moderate lipophilicity. Compound 5i could
serve as a useful chemical probe to help establish a proof-of-concept of OGA inhibition as a therapeutic target for the treatment of
tauopathies.
increasing attention as a potential therapeutic target for the
treatment of tau-related neurodegenerative diseases.
INTRODUCTION
■
Aggregation of the tau protein is one of the hallmarks of the
progression of neurodegenerative disorders, such as Alz-
heimer’s disease, Pick’s disease, and Parkinson’s disease.1,2
Results from recently published studies indicate that O-
GlcNAcylation, which consists of the glycosylation of serine or
threonine residues with O-linked N-acetylglucosamine (O-
GlcNAc), plays an important role in aggregation of the tau
protein.3,4 O-GlcNAcylated tau protein displays improved
stability and solubility compared with its unmodified state,
which in turn leads to a suppression of the aggregation of the
tau protein. The results of some clinical studies indicated that
the O-GlcNAcylation level of tau protein decreases in patients
with Alzheimer’s disease.5,6 Notably, O-GlcNAcylation is
regulated by two enzymes: O-GlcNAc transferase (OGT)
and O-GlcNAcase (OGA).7,8 OGT catalyzes the addition of O-
GlcNAc to protein substrates, whereas OGA catalyzes the
cleavage of O-GlcNAc from the O-glycosylated proteins. Since
inhibition of OGA has been reported to increase the level of O-
GlcNAcylation of tau proteins, OGA has been the object of
OGA consists of two domains: a C-terminal domain
displaying histone acetyltransferase activity and an N-terminal
domain displaying O-GlcNAcase activity. The N-terminal
domain, which is classified into the glycoside hydrolase family
84 (GH84),9 breaks the glycosidic bond of the O-GlcNAc
group attached to proteins. Crystal structures of OGA in
complex with substrate analogues acting as enzyme inhibitors
determined by X-ray diffraction have been reported over the
past few decades. These structures enabled us to understand
the interactions between OGA and its protein substrates as
well as the catalytic mechanism of the hydrolysis of O-
Received: September 29, 2020
Published: January 6, 2021
https://dx.doi.org/10.1021/acs.jmedchem.0c01712
© 2021 American Chemical Society
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Figure 1. Reported inhibitors of O-GlcNAcase (OGA). Crystal structures of the various compounds in complex with bacterial OGAs (human and
bacterial in the case of inhibitor 1a) are indicated, with their corresponding letter designations specified in brackets.
glycosylated proteins.10−12 O-GlcNAcylated proteins bind to
OGA through hydrogen bonds with polar residues such as
Asp285 and Asn313. The residue pair of Asp174 and Asp175
assists the hydrolysis reaction by donating and accepting
electrons and protons with the substrate.
active compounds to be identified with a high hit rate because
this type of method explores compound libraries focusing on
common chemical features or pharmacophores with known
active compounds, based on the fact that compounds
displaying structural similarities with respect to the mentioned
features often have similar biological profiles. A structure-based
approach sequentially added to the described ligand-based
approach could be a powerful method for identifying
potentially active compounds for unfavorable contact with
amino acids or quantitated binding affinity to the target
protein. Therefore, we applied a multiple hit exploration
approach combining a structure-based method and a ligand-
based one. In the first step of the approach, hit compounds
were extracted by ligand-shape-based virtual screening, and
thus, the obtained compounds were subsequently narrowed
down by conducting a sequential docking study. Furthermore,
filtering of the compounds by physicochemical parameters for
druglike properties and central nervous system (CNS)
penetration was incorporated in the approach to enrich the
qualities of a compound set to be examined in biological
assays. The identified hit compounds were optimized to
improve their pharmacokinetic profile focusing on ligand
lipophilicity efficiency (LLE).25−27 By this approach, we were
able to identify a brain-penetrable in vivo tool compound. We
hereby report the discovery of a novel in vivo tool compound
starting from hit identification achieved by computer-aided
virtual screening and subsequent hit/lead optimization.
Based on the structural insights thus achieved, several OGA
inhibitors have been discovered to date, with some
representative compounds depicted in Figure 1.13−18 Notably,
most of these inhibitors possess sugar-like substructures that
mimic the natural substrate of the enzyme, O-GlcNAc. As
these compounds comprise a number of hydroxy groups in
their sugar-like scaffold, these inhibitors might exhibit a high
polarity, which might be a concern about low brain
penetration. In 2019, a sugar-mimetic OGA inhibitor MK-
8719, with improved pharmacokinetic profile and brain
penetration, was reported as a compound to be tested in a
clinical trial;19 however, non-sugar-like compounds with high
potency and better pharmacokinetic profiles are still awaited to
help characterize OGA.
As X-ray crystal structures of OGA in complex with several
OGA inhibitors have been reported, hit compounds can be
explored by virtual screening efficiently.20 Structure-based
virtual screening using crystal structures has been widely
performed;21 in fact, a study of hit discovery for OGA by
docking-based virtual screening was published in 2019.22 In it,
multiple docking-based methods with different accuracy levels
were implemented sequentially, resulting in the identification
of a non-sugar-type OGA inhibitor. However, this compound
exhibited lower OGA inhibitory potency than traditional sugar-
type inhibitors, which suggests that the development of a new
strategy could be beneficial to the search for potent and novel
OGA inhibitors. Indeed, known OGA inhibitors allow us to
implement a ligand-based approach, such as similarity search
for analogues or bioisosteres.23 When three-dimensional (3D)
conformations of query compounds are determined or
predicted, pharmacophore search or shape similarity search
can also be applied.24 Ligand-based virtual screening allows
RESULTS AND DISCUSSION
■
Homology Modeling. Although about 20 X-ray crystal
structures of human OGA have been reported so far,12,19,28
only the crystal structures of OGAs from Bacteroides
thetaiotaomicron (BT_4395), Clostridium perfringens, and
Oceanicola granulosus had been published at the time of the
present study.13−18,29 Among these three bacterial proteins, the
amino acid sequence of BT_4395 has the highest identity with
the sequence of human OGA. Thus, BT_4395 was selected for
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Figure 2. Alignment of the amino acid sequences between human O-GlcNAcase (OGA) and bacterial BT_4395 OGA. Amino acid residues known
to be within 8 Å of the ligand Thiamet-G in the crystal structures of the complexes between human OGA and the said substrate analogue and
BT_4395 OGA and the same ligand are colored in red; moreover, the residues involved in direct interactions with Thiamet-G are highlighted in
yellow.
Figure 3. Interactions between BT_4395 O-GlcNAcase (OGA) and Thiamet-G (PDB ID: 2VVN). Hydrogen bonds are indicated by red dashed
lines. Amino acid residues interacting directly with Thiamet-G are made evident by green-colored thick sticks and their letter designations are
highlighted in yellow boxes. Amino acid residues that are not conserved between BT_4395 OGA and human OGA are indicated by magenta-
colored stick, with their letter designations highlighted in cyan boxes.
sequence and structure analysis to compare with human OGA.
Notably, the amino acid identity between human OGA and
BT_4395 OGA in the catalytic domain in N-terminal domains
of these proteins was 33%, whereas the X-ray crystal structure
of BT_4395 OGA in complex with an OGA inhibitor,
Thiamet-G (PDB code: 2VVN13), indicated an approximately
70% conservation of amino acid residues within 8 Å from
Thiamet-G (Figure 2). Furthermore, all of the seven amino
acid residues interacting with Thiamet-G are identical between
human and BT_4395 OGA: Asp174, Asp285, Asn313, Gly67,
Lys98, Tyr219, and Trp278 (human OGA numbering). The
major differences around the ligand binding sites are Trp286
(BT_4395 OGA)/Phe223 (human OGA) and His433
(BT_4395 OGA)/Tyr569 (human OGA) (Figure 3). Since
these residues are located around the entrance to the substrate-
binding pocket and the values for their B-factors are higher
than those of other residues, the residues specified above
should only make a small contribution to protein−ligand
binding. This speculation is supported by the crystal structures
of OGA in complex with many potent inhibitors, which bind to
the catalytic site in the absence of interactions with Trp286
and His443. Therefore, a homology model of human OGA
based on the structure of bacterial BT_4395 OGA is applicable
with enough accuracy for docking-based virtual screening.
Thus, we constructed a homology model of human OGA using
the crystal structure of BT_4395 OGA in complex with
Thiamet-G (PDB code: 2VVN) as a template. In fact, by
superposing and comparing the structure of BT_4395 OGA in
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complex with Thiamet-G (PDB code: 2VVN14) to that of
human OGA in complex with Thiamet-G (PDB code:
5M7S28), it can be concluded that not only the backbones
of the two proteins but also the side chains around the ligand
binding sites adopted essentially identical conformations.
Virtual Screening. As mentioned above, a number of
sugar-derived OGA inhibitors are currently known. However,
their high polarity might be a concern from the standpoint of
pharmacokinetics. To estimate the possibility of identifying hit
compounds with moderate lipophilicity by virtual screening, a
protein druggability assessment was carried out to examine the
profile of the ligand binding site of OGA using SiteMap30,31
module in Maestro.32 SiteMap computes SiteScore and DScore
values in terms of the size, tightness, and hydrophobic and
hydrophilic characters of a pocket. The values of SiteScore and
DScore for the said pocket in the structure of the complex
between BT_4395 OGA and Thiamet-G (PDB code: 2VVN)
were 1.15 and 1.20, respectively, which suggested that OGA
has a highly druggable pocket. Therefore, a hit exploration
strategy starting from a ligand-based approach followed by the
reranking of the deliverables by a sequential docking-based
approach could identify high-quality hit compounds with lead-
like properties.
structures, OGA inhibitors form hydrogen bonds with polar
amino acids, such as Asp285, Asp174, or Asn313. This
evidence led us to pick out 70 000 compounds that formed
hydrogen bonds with at least one residue among the
mentioned three. Finally, to focus on compounds with CNS-
drug likeness, the extracted compounds were filtered based on
the range of physicochemical properties used to define the
CNS multiparameter optimization (CNS-MPO) index35
(molecular weight ≤ 350, A log P ≤ 5, number of hydrogen
donors ≤ 4, and topological polar surface area ≤ 125) and the
indicators of lead likeness that are not included in the CNS-
MPO index (number of hydrogen acceptors ≤ 8 and number
of rotatable bond ≤ 6); by this process, 55 000 compounds
were retained. This set of compounds was subsequently filtered
applying the pan assay interference compound (PAINS) filter.
To submit a diverse compound set to our biological assay of
choice, the compounds were divided into 2681 clusters based
on molecular similarity and a total of 2681 compounds, the
top-ranking compounds of each cluster, were subjected to a
biological assay. The workflow of the virtual screening is
depicted in Figure 4.
The crystal structures of the complexes between OGA and
several known OGA inhibitors have been reported over the
past decade. The active conformations of these ligands, as
determined through the said crystal structures, highlight the
steric and electrostatic features of the ligand that induce OGA
inhibition. Therefore, a 3D ligand-based approach utilizing
these active conformations was applied as the first step of the
hit exploration process. Multiple ligands used as queries of the
ligand-based approach could expand the chemical space to be
explored. We selected six inhibitors, compounds 1a−f, that
exhibited high potency and for which the crystal structure of
their complexes with OGA were available (Figure 1). Although
the crystal structures of human OGA had not yet been
published at the time of the present study, it was speculated
that there was only small difference between the protein
conformations around the active site of human OGA and
bacterial OGA according to structural comparison analysis
mentioned in the previous section, which indicated that the
bound conformations of the ligands are almost identical.
Therefore, we used the coordinates from the crystal structures
in complex with bacterial OGA. The 3D ligand-based
approaches mainly fall into two categories: the molecular
shape similarity-based approach and the pharmacophore-based
approach. Given that the reported OGA inhibitors have
characteristic sp3-rich frameworks and bind to the pocket via
several hydrogen bonds with amino acid residues, both the
shape and the chemical characteristics of a molecule would be
important factors that obtain binding affinity of the said
molecule to OGA. Based on the fact that the utilization of
ROCS, one of standard methods for shape similarity searches,
yields overlay patterns whereby not only the shape of the
molecule but also their chemical features are matched,33,34
ROCS was used for ligand-based virtual screening.
Figure 4. Workflow of virtual screening.
Structure-Based Analysis of Hit Compound. The OGA
inhibitory activities of the 2681 compounds were measured
using the full-length human OGA protein and 4-methyl-
umbelliferyl N-acetyl-β-D-glucosaminide dihydrate as fluoro-
genic substrate. The initial screening at 10 μM concentration
led to the identification of 13 compounds displaying more than
50% inhibition. The subsequent dose−response assay allowed
us to identify compound 2 (Figures 5a and S1), which
exhibited a potent inhibitory activity with a measured IC50
value of 41 nM. Compound 2 was confirmed to exhibit target-
specific inhibitory activity toward OGA via performance of a
counter-assay involving β-hexosaminidase, which, similar to
OGA, catalyzes the hydrolysis of O-glycosylated proteins.
Notably, 2 did not exhibit any inhibitory activity against β-
hexosaminidase at 10 μM concentration. To begin lead
optimization utilizing the structure-based drug design, the
docking model of compound 2 was constructed (Figure 5b).
The binding mode obtained in the virtual screening was
carefully checked to compare each generated binding pose of
all of the stereoisomers, R-form and S-form, and protonation
states that exist in a possible pH range. Notably, the pyrimidine
In the first stage of the virtual screening, a 3D shape
similarity search of a corporate library was carried out against
six query compounds using ROCS. Subsequently, we
conducted the docking of approximately 100 000 compounds
selected by 3D molecular shape to prioritize compounds
exhibiting favorable contacts or forming additional interactions
with amino acid residues. According to the published crystal
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Figure 5. (a) Chemical structure of compound 2. (b) Docking model of 2 in S-configuration into human O-GlcNAcase (OGA) homology model
and WaterMap analysis for the docked protein. The most stable water molecules, which exhibit the lowest ΔG values, are colored in green, and the
most unstable water molecules, which exhibit the highest ΔG values, are colored in red. The other water molecules are color-coded from green to
red by gradation according to their ΔG values. Notably, the unstable water molecules are labeled as 1, 2, and 3, and the stable ones are labeled as 1′,
2′, 3′, and 4′. (c) Enthalpy, entropy, and free-energy values for water molecules as computed by WaterMap.
and the piperazine rings could exist in three and two distinct
possible protonation states, respectively: the cationic states
whereby the N-1 or the N-3 positions are protonated and the
neutral state, for the pyrimidine; the cationic states whereby
the N-1′ or the N-4′ positions are protonated, for the
piperazine. The pKa values of the nitrogen atoms are 4.9 for
N(1) and 6.7 for N(3) in the case of the pyrimidine ring and
8.2 for N(1′) and 8.4 for N(4′) in the case of the piperazine
ring.32,36 As the acidity might be relatively high around the
pyrimidine ring due to the presence of the acidic amino acids,
Asp285 and Asp174, in the active site of OGA, even the
protonated form of N(1) could exist in a stable form in this
specific region. Based on the results of the docking study
performed taking into consideration all possible combinations
of the two stereoisomers and various protonation states by
LigPrep,32 the S-isomer of 2, whereby N(1) and N(4′) were
protonated, exhibited the most reasonable binding mode. In
this docking mode, the N(1) of the pyrimidine and the
neighboring amine form a bidentate hydrogen bond with the
carboxyl acid group of Asp285. The methylpiperidine group
binds to the deep pocket stacking with Tyr219 and Trp278.
The piperazine group is positioned at the entrance of the
ligand binding site, where Val255 and Tyr286 are located,
without getting involved in direct interactions with any amino
acid residues.
protein and water [#HB(PW)], so that it provides
physicochemical insight of the binding site.
The predicted thermodynamic parameters of the water
molecules are reported in Figure 5b,c. According to the result
of WaterMap calculations, the three most unstable water
molecules, 1, 2, and 3, with ΔG values in the range of 4−8
kcal/mol gather in the deep site consisting of Tyr219 and
Trp278. Since the displacement of unstable water molecules to
the bulk solution leads to a gain in free energy, this deep site
makes a large contribution to the protein−ligand binding. As
speculated by the surrounding hydrophobic side chains of
Tyr219 and Trp278, SiteMap also suggested this site to be
hydrophobic; this site is mainly covered with the hydrophobic
surface in the surface map drawn by SiteMap (Figure S2). In
spite of the presence of the said hydrophobic pocket, the
#HB(PW) values of the three water molecules ranged from 0.5
to 0.9, which means that these water molecules were on
average engaged in 0.5−0.9 hydrogen bond interactions during
the simulation. The oxygen atom in the main chain of Pro216,
which is slightly facing the pocket surface, could be a hydrogen
bond counter at part of simulation time. Although the deep
hydrophobic pocket displays a tendency to prefer hydrophobic
substituents of the ligand, the properties of the water molecules
as computed by WaterMap indicate that a heteroatom is also
acceptable.
On the other hand, the four most stable water molecules
with ΔG values lower than −1 kcal/mol are observed near
Asp285. The water molecules in this site show a large enthalpy
gain (ΔH ranged from −7 to −2 kcal/mol); moreover, here
enthalpy is related mainly to nonbonding interaction. The
water molecules 1′ and 4′ (Figure 5b,c) form hydrogen bonds
with Asp285. Since the nitrogen atoms of the aminopyrimidine
group overlap well with these water molecules, the amino-
pyrimidine group is predicted to find itself in the proper
position to form hydrogen bonds with Asp285.
To elucidate the properties of the ligand binding site and the
protein−ligand interaction in more detail, a WaterMap
calculation was applied to the docking model of compound
2 with the hOGA homology model.37,38 In this approach,
water molecules present in a ligand binding site of the protein
are displaced and their free-energy values are computed.
WaterMap provides not only the values for enthalpy (ΔH) and
entropy (−TΔS), which are the components of free energy
(ΔG), but also the number of hydrogen bonds between
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ab
Scheme 1. Synthetic Route of Pyrimidine Derivatives
a
b
Amines, triethylamine, tetrahydrofuran (THF), room temperature. Amines, N,N-diisopropylethylamine (DIPEA), solvents, heating.
Table 1. Inhibitory Activities of Compounds 2 and 5a against OGA
a
IC50 values and 95% confidence intervals (given in parentheses) were analyzed by duplicate measurements with GraphPad Prism V.5 (GraphPad
Software, San Diego, CA) statistical analysis software using a nonlinear regression for a sigmoidal dose−response curve with a variable slope.
Water molecules exhibiting moderate values for the free
energy (ΔG = −1−1 kcal/mol) were observed around the
residues, Val255 and Tyr286, at the entrance of the pocket.
This observation indicates that the said entrance is a solvent-
accessible region, which allows various substituents. The
methyl group attached to the chiral carbon also seems to
engage in no specific interactions with amino acids. Consistent
with this observation, no unstable water molecules were
located in the position of the methyl group, supporting the
speculation that this methyl group made no significant
contribution to binding affinity. Therefore, this methyl group
could be removed from compound 2, with the ligand
maintaining its inhibitory potency toward OGA.
Chemistry. The designed compounds were synthesized
according to Scheme 1. 2,4-Diaminopyrimidines 5a−i were
synthesized by the sequential nucleophilic substitution of 2,4-
dichloropyrimidine 3 and corresponding amines (Scheme 1).
Biological Evaluation. To initiate structure−activity
relationship (SAR) studies, a chemically accessible scaffold
would be preferable. WaterMap analysis results, reported in
Figure 5b,c, suggested that the piperazine group might be
replaced by various substituents, with little influence on the
affinity. Replacement of the piperazine group with a phenethyl
group produced compound 5a, which retained the inhibitory
potency of compound 2 toward OGA (IC50 = 24 nM; Table
1). This simple compound was a good starting point for further
optimization. To characterize the inhibitory mechanism of the
chemical series, a kinetic assay of 5a was carried out. The
substrate-competitive profile of 5a was investigated by
estimating the change in Km values as a result of changes in
compound concentration. As can be observed from Figure S3,
increase in the Km value was associated with increase in 5a
concentration, indicating that 5a inhibits the OGA activity in a
substrate-competitive manner.
Ligand lipophilicity efficiency (LLE) was considered during
further optimization. Notably, LLE has been reported to be a
useful concept in lead discovery and optimization to make
more specific interactions with a target molecule.25−27
Compound 5a exhibited a moderate LLE value of 3.5 due to
its relatively high lipophilicity (A log P39,40 = 4.2). We explored
the substituent targeting the deep pocket of OGA (Table 2).
Although the results of the WaterMap analysis suggested the
deep pocket to be mostly hydrophobic, the opportunity still
seems to exist for the said pocket to accommodate a
hydrophilic substituent (Figure 5b,c). To reduce the lip-
ophilicity of the ligand, 6-membered cyclic amines were
introduced, such as morpholine (5b; LLE = 2.7) and
methylpiperazine (5c; IC50 > 10 000 nM), that had decreased
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b
Table 2. OGA Inhibitory Activities and Ligand Lipophilic Efficiency of Compounds 5a−i
a
IC50 values and 95% confidence intervals (given in parentheses) were analyzed by duplicate measurements with GraphPad Prism V.5 (GraphPad
b
Software, San Diego, CA) statistical analysis software using a nonlinear regression for a sigmoidal dose−response with a variable slope. LLE =
pIC50 − A log P.
LLE. Further modification of the piperidine was explored by
introducing polar substituents. The introduction of a hydroxy
or a methoxy group produced no beneficial effects (4-
hydroxypiperidine 5d; IC50 > 10 000 nM, 4-methoxypiperidine
5e; LLE = 3.2). Improving the LLE by introducing a polar
substituent targeting the deep pocket proved difficult to
achieve. On the other hand, various modifications could be
applied to the phenethyl group, as suggested by the WaterMap
calculation data and inhibitory activities of 2 and 5a. To
improve the LLE value, the phenyl ring of 5a was replaced with
a pyridine ring, which is characterized by lower lipophilicity.
The 2-pyridine derivative of 5a (5f; LLE = 3.3) had a similar
LLE value to 5a, whereas the 3-pyridine derivative (5g; LLE =
4.5) displayed an increased LLE value. The corresponding
methylene derivative 5h (LLE = 5.1) was characterized by an
even higher value of LLE and a lower value of A log P. Further
modification with nitrogen-containing heterocycles yielded
compound 5i (IC50 = 46 nM; LLE = 5.2), which had the
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characteristics of a potent OGA inhibitor with a desirable LLE
value. The CNS-MPO values of the mentioned compounds
were also monitored throughout the optimization. Although
the lead compound 5a was characterized by a modest CNS-
MPO value, due to its high lipophilicity, the value of CNS-
MPO was observed to increase mainly as a result of the
decrease of the A log P value; ultimately, 5i was observed to
display a substantial increase in the CNS-MPO value.
A detailed characterization of compound 5i was conducted
in terms of its efficacy in cells, its selectivity, and its
pharmacokinetic profile. The levels of O-GlcNAcylated protein
were evaluated by in-cell Western assay. Compound 5i induced
glycosylation in cells with an EC50 value of 450 nM for causing
an increase of O-GlcNAcylated protein to half-maximal level
(95% confidence intervals = 190−1100 nM, Emax = 143%). In
terms of the assessment of selectivity, the in vitro inhibitory
activity of 5i was evaluated against β-hexosaminidase and
various kinases. In detail, compound 5i did not display any
folded conformation that is essentially identical to those of the
other reported structures of bacterial OGA. Compound 5a
mostly displays a common protein−ligand interaction pattern
in the crystal structure compared with that of the human OGA
homology model; the aminopyrimidine core was involved in a
bidentate hydrogen bonds with Asp344 (corresponding to
Asp285 in human OGA). However, one exception was found;
a difference between the two structures lies in the
conformation of Asp242 (corresponding to Asp174 in
human OGA), which is located around the gate region of
the deep pocket. Not only the methylpiperidine group bound
to the deep pocket stacks with the side chains of Tyr282 and
Trp337 (corresponding to Tyr219 and Trp278 in human
OGA) as the docking model suggested, but it also kicks out
Asp242. Although most reported OGA inhibitors like Thiamet-
G were observed to pull in Asp242 to form hydrogen bonds
with this residue in the gate region of the deep pocket, the
crystal structure of the complex between BT_4395 OGA and
compound 5a suggested that 5a engaged in a tight hydro-
phobic interaction with the protein by causing the methyl-
piperidine group to be placed at the bottom of the deep pocket
kicking out Asp242. By utilizing the characteristic hydrophobic
interaction, this compound achieved high affinity with OGA
despite the reduced number of hydrogen bond donors and the
moderate lipophilicity. Notably, an intramolecular π−π
stacking between the pyrimidine core and the terminal
benzene ring was also observed in the mentioned crystal
structure. This stacking interaction helped in keeping 5a in a
rigid conformation. Replacement of the benzene ring with the
pyridine ring (see 5f and 5g), which is characterized by lower
π-electron density, reduced the π−π stacking effect.42 In
contrast, incorporation of 3-pyridine to produce 5g led to the
retention of the potency of the inhibitor, possibly because the
nitrogen atom at the 3-position of the pyridine ring might
interact with the adjacent Tyr219 in human OGA via a water-
mediated hydrogen bond. Although the predicted docking
model was in good agreement with the experimentally
determined structure, with respect to protein−ligand inter-
actions, the crystal structure suggested a more detailed
significant inhibitory activity against β-hexosaminidase (IC50
>
10 μM) and 277 kinases (IC50 > 1μM) under the assay
condition described previously (Figure S4).41
Finally, a pharmacokinetic study of compound 5i was
performed in mice. The high exposure levels of 5i were
observed in the plasma and the brain at the 0.5 and 1 h marks
after oral administration to mice (Table 3).
Table 3. Pharmacokinetic Properties of 5i in the Plasma and
Brain of Mouse
a
time (h)
Cplasma (μg/mL)
Cbrain (μg/g)
Kp,uu
0.5
1.0
2.8
1.9
2.7
2.6
0.25
0.36
a
Brain-to-plasma unbound concentration ratio (Kp,uu) calculated from
the values for the unbound fraction in plasma (fu,plasma) and brain
(fu,brain) of 0.53 and 0.14, respectively.
Crystal Structure of Compound 5a. The structure of
compound 5a bound to BT_4395 OGA was determined by X-
ray crystallography (Figure 6a). The protein structure adopts a
Figure 6. (a) Crystal structures of the complexes between BT_4395 O-GlcNAcase (OGA; green) and compound 5a (yellow) overlaid with the
crystal structure (white) of BT_4395 OGA in complex with Thiamet-G (Thiamet-G not shown; PDB code 2VVN), and (b) docking model of 5i
(cyan) into the human OGA homology model (green). Hydrogen bonds are represented by red dashed lines. CH−π or π−π interactions are
identified by red arrows.
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interpretation for the SAR of the terminal aryl ring, which was
attributed to the intramolecular π−π stacking.
combining ligand-based and structure-based approaches
followed by optimization focusing on LLE. Compound 5i
exhibited efficacy in cells and a favorable pharmacokinetic
profile with brain penetration. The OGA inhibitor 5i could be
a useful in vivo chemical tool to elucidate the function of OGA
in tauopathies and neurodegenerative events, and it has the
potential to be developed as a therapeutic agent in the
treatment of tau-mediated neurodegenerative diseases.
Docking Model of Compound 5i. To interpret the SAR
and high LLE of compound 5i, the docking model of 5i was
constructed based on the crystal structure of 5a in complex
with BT_4395 OGA (Figure 6b). In addition to the common
interactions with Asp285, Tyr219, and Trp278 (corresponding
to Asp344, Tyr282, and Trp337, respectively, in BT_4395
OGA) observed for 5a, the terminal methylimidazole moiety
was predicted to engage in CH−π interactions with Val254
and Tyr286 at the entrance of the pocket. Due to the
decreased length of the alkyl linker compared with that of the
original compound 5a, intramolecular π−π stacking involving
the terminal aryl ring should be lost; however, favorable
contacts with Val254 and Tyr286 should be gained, so that the
inhibitory potency toward OGA is retained. To confirm the
stability of the predicted binding mode and pivotal
interactions, a molecular dynamics (MD) simulation of 5i in
complex with the human OGA homology model was carried
out. The binding mode was found to be stable during the MD
simulation since the root-mean-square deviation values of the
protein backbone and the heavy atoms of the ligand were kept
in the range of 2.8−3.2 Å and 1.2−2.4 Å, respectively (Figure
S5a). As can be observed from the interaction diagram during
the MD simulation, hydrogen bonds were observed to exist
between the protonated pyrimidine and Asp285 throughout
the entire duration of the simulation (Figure S5b). The
hydrogen bonds were inferred to be pivotal for binding to
OGA. Furthermore, the MD simulation revealed an interaction
with Tyr69, which might arise as a byproduct of hydrogen-
bonding interactions between Asp285 and the nitrogen atoms
of the pyrimidine ring. Tyr69 was located 4.8 Å from the
center of the pyrimidine ring. Although the two aromatic rings,
the phenol ring of Tyr69 and the pyrimidine ring of 5i, are too
distant from each other to engage in ideal π−π interactions, the
hydrogen bonds to Asp285 fixed the position of the pyrimidine
ring and helped to create the interaction with Tyr69 over a
long time. Consistently with the insight afforded by the
docking model, the MD simulation supported the existence of
π−π stacking interactions of 5i with Tyr219 and Trp278 and
CH−π interactions with Val254 and Tyr286.
EXPERIMENTAL SECTION
Chemistry: General. H NMR spectra were obtained at Bruker
■
1
AVANCE 300 (300 MHz). Chemical shifts are given in δ values
(ppm) using tetramethylsilane as the internal standard. Peak
multiplicities are expressed as follows: s, singlet; d, doublet; t, triplet;
dd, doublet of doublets; tt, triplet of triplets; ddd, doublet of doublet
of doublets; brs, broad singlet; m, multiplet. Reaction progress was
determined by thin-layer chromatography (TLC) analysis on Merck
Kieselgel 60 F254 plates or Fuji Silysia NH plates. Chromatographic
purification was carried out on silica gel columns (Merck Kieselgel 60,
70−230 mesh, or 230−400 mesh, Merck; Chromatorex NH-DM
1020, 100−200 mesh, Fuji Silysia Chemical; Inject column and
Universal column, YAMAZEN; or Purif-Pack Si or NH, Shoko
Scientific). Low-resolution mass spectra (MS) were acquired using an
Agilent LC/MS system (Agilent1200SL/Agilent6130MS) or Shimad-
zu UFLC/MS (Prominence UFLC high-pressure gradient system/
LCMS-2020) operating in an electrospray ionization mode (ESI+).
The column used was an L-column 2 ODS (3.0 mm × 50 mm ID, 3
μm, CERI) with a temperature of 40 °C and a flow rate of 1.2 or 1.5
mL/min. Condition 1: mobile phases A and B under acidic conditions
were 0.05% trifluoroacetic acid (TFA) in water and 0.05% TFA in
MeCN, respectively. The ratio of mobile phase B was increased
linearly from 5 to 90% over 0.9 min, 90% over the next 1.1 min.
Condition 2: mobile phases A and B under neutral conditions were a
mixture of 5 mM AcONH4 and MeCN (9/1, v/v) and a mixture of 5
mM AcONH4 and MeCN (1/9, v/v), respectively. The ratio of
mobile phase B was increased linearly from 5 to 90% over 0.9 min,
90% over the next 1.1 min. Chemical intermediates were
characterized by 1H NMR or mass spectral data or both. The purities
of all tested compounds in biological systems were confirmed to be
more than 95% purity as determined by analytical high-performance
liquid chromatography (HPLC). Analytical HPLC was carried out
using a Corona CAD (charged aerosol detector) or a photodiode
array detector. The column was a Capcell Pak C18AQ (50 mm × 3.0
mm ID, Shiseido, Japan) or an L-column 2 ODS (30 mm × 2.0 mm
ID, CERI, Japan) with a temperature of 50 °C and a flow rate of 0.5
mL/min. Mobile phases A and B under neutral conditions were a
mixture of 50 mM AcONH4, water, and MeCN (1:8:1, v/v/v) and a
mixture of 50 mM AcONH4 and MeCN (1:9, v/v), respectively. The
ratio of mobile phase B was increased linearly from 5 to 95% over 3
min, 95% over the next 1 min. Mobile phases A and B under acidic
conditions were a mixture of 0.2% formic acid in 10 mM ammonium
formate and 0.2% formic acid in MeCN, respectively. The ratio of
mobile phase B was increased linearly from 14 to 86% over 3 min,
86% over the next 1 min. Yields refer to isolated and purified products
derived from nonoptimized procedures. Reagents and solvents were
obtained from commercial sources and used without further
purification.
Since polar amino acids such as Asp174 and Asp285 are
located in the active site of OGA, the reported hydrophilic
sugar-derived OGA inhibitors bind to the active site mainly via
polar interactions. On the other hand, compound 5i, with its
moderate lipophilicity, achieved potent OGA inhibition
through tight binding with the hydrophobic deep pocket
comprising Tyr219 and Trp278 with kicking out of Asp174
and CH−π interaction with Val254 and Tyr286, which are
located at the entrance of the pocket. Furthermore, the
aminopyrimidine scaffold, which would be in its stable neutral
form under physiological conditions (pH ∼ 7.4), would be
protonated at the nitrogen atom in the specific environment,
where it is surrounded by several Asp residues, and thus, it
would act as a hydrogen bond donor. The superior brain
penetration of compound 5i would be achieved by the
aminopyrimidine core structure engaging in a crucial
interaction with Asp285 accompanying a reduced number of
hydrogen bond donors.
Structural Characterization of Hit Compound (2). 1H NMR (300
MHz, DMSO-d6) δ 0.85−1.12 (8H, m), 1.54−1.71 (3H, m), 2.05−
2.48 (13H, m), 2.68−2.86 (2H, m), 3.90−4.08 (1H, m), 4.18−4.36
(2H, m), 5.97 (1H, d, J = 6.0 Hz), 6.06 (1H, d, J = 7.2 Hz), 7.73 (1H,
d, J = 6.0 Hz). MS: [M + H]+ 333.3. HPLC purity 91%.
General Procedure A: Combinatorial Nucleophilic Substitution
Reaction of Compound (4a). The mixture of compound 4a (0.017 g,
0.08 mmol), corresponding amine (0.120 mmol), DIPEA (0.042 mL,
0.240 mmol), and dimethyl sulfoxide (DMSO, 1 mL) was heated at
150 °C for 1 h under microwave irradiation. The reaction mixture was
diluted with MeCN (0.5 mL) and purified by preparative HPLC
(Actus Triart C18, eluted with MeCN/0.1% TFA aqueous solution).
CONCLUSIONS
We have discovered a novel OGA inhibitor, compound 5i,
utilizing virtual screening based on multiple methodologies
■
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Pure fractions were combined and concentrated by blowing away with
the air at 60 °C to yield a product.
4-(4-Methylpiperidin-1-yl)-N-(pyridin-3-ylmethyl)pyrimidin-2-
amine (5h). To a mixture of compound 4a (0.17 g, 0.80 mmol) in
tert-AmOH (1.606 mL) were added 3-picolylamine (0.082 mL, 0.80
mmol) and DIPEA (0.421 mL, 2.41 mmol) at room temperature. The
mixture was heated to 150 °C for 8 h under microwave irradiation in a
sealed tube. The mixture was concentrated in vacuo and purified by
column chromatography (NH silica gel, eluted with 40−100% EtOAc
in hexane and 0−20% MeOH in EtOAc) to yield compound 5h
(0.090 g, 40%) as a white solid after recrystallization from EtOAc/
General Procedure B: Combinatorial Nucleophilic Substitution
Reaction of Compound (4b). The mixture of compound 4b (0.019 g,
0.08 mmol), corresponding amine (0.160 mmol), DIPEA (0.070 mL,
0.400 mmol), and tert-AmOH (1 mL) was heated at 130 °C for 1 h
under microwave irradiation. The reaction mixture was diluted with
MeCN (0.5 mL). The mixture was purified by preparative HPLC
(Actus Triart C18, eluted with MeCN/0.1% TFA aqueous solution).
Pure fractions were combined and concentrated by blowing away with
the air at 60 °C to yield a product.
2-Chloro-4-(4-methylpiperidin-1-yl)pyrimidine (4a). To a solu-
tion of 2,4-dichloropyrimidine (8.15 g, 54.71 mmol) and 4-
methylpiperidine (7.77 mL, 65.65 mmol) in THF (300 mL) was
added triethylamine (11.41 mL, 82.06 mmol) at room temperature.
The mixture was stirred at room temperature over the weekend. The
mixture was quenched with water at room temperature and extracted
with EtOAc. The organic layer was separated, washed with brine,
dried over Na2SO4, and concentrated in vacuo. The residue was
purified by column chromatography (silica gel, eluted with 40−100%
EtOAc in hexane) to yield compound 4a (9.09 g, 78%) as colorless
crystals. 1H NMR (300 MHz, DMSO-d6) δ 0.91 (3H, d, J = 6.0 Hz),
0.95−1.13 (2H, m), 1.58−1.76 (3H, m), 2.91 (2H, t, J = 12.2 Hz),
4.30 (2H, brs), 6.82 (1H, d, J = 6.0 Hz), 8.01 (1H, d, J = 6.4 Hz). MS:
[M + H]+ 212.1.
1
hexane. H NMR (300 MHz, DMSO-d6) δ 0.81−1.06 (5H, m), 1.60
(3H, d, J = 10.2 Hz), 2.66−2.83 (2H, m), 4.24 (2H, d, J = 13.2 Hz),
4.41 (2H, d, J = 6.2 Hz), 6.01 (1H, d, J = 6.2 Hz), 7.09 (1H, brs),
7.30 (1H, dd, J = 7.7, 4.7 Hz), 7.68 (1H, dd, J = 10.0, 1.7 Hz), 7.75
(1H, d, J = 6.0 Hz), 8.39 (1H, dd, J = 4.7, 1.7 Hz), 8.51 (1H, d, J =
1.7 Hz). MS: [M + H]+ 284.3.
N-((1-Methyl-1H-imidazol-4-yl)methyl)-4-(4-methylpiperidin-1-
yl)pyrimidin-2-amine (5i). To a solution of (1-methyl-1H-imidazol-4-
yl)methylamine (50 mg, 0.45 mmol) and compound 4a (105 mg, 0.49
mmol) in tert-AmOH (3 mL) was added DIPEA (0.775 mL, 4.50
mmol) at room temperature. The mixture was sealed and stirred at
130 °C overnight. The mixture was concentrated in vacuo. The
residue was purified by column chromatography (NH silica gel, eluted
with 50−100% EtOAc in hexane and 0−20% MeOH in EtOAc) to
1
yield compound 5i (55.0 mg, 43%) as colorless crystals. H NMR
(300 MHz, CDCl3) δ 0.95 (3H, d, J = 6.0 Hz), 1.04−1.19 (2H, m),
1.51−1.65 (2H, m), 1.70 (1H, brs), 2.71−2.85 (2H, m), 3.63 (3H, s),
4.33 (2H, d, J = 12.8 Hz), 4.52 (2H, d, J = 5.7 Hz), 5.12 (1H, brs),
5.88 (1H, d, J = 6.4 Hz), 6.79 (1H, s), 7.34 (1H, s), 7.86 (1H, d, J =
6.4 Hz). MS: [M + H]+ 287.2.
4-Chloro-N-phenethylpyrimidin-2-amine (4b). To a solution of
2,4-dichloropyrimidine (10 g, 67.12 mmol) and phenethylamine
(12.64 mL, 100.69 mmol) in THF (250 mL) was added triethylamine
(18.66 mL, 134.25 mmol) at room temperature. The mixture was
stirred at room temperature for 5 h. The mixture was quenched with
water at room temperature and extracted with EtOAc. The organic
layer was separated, washed with brine, dried over Na2SO4, and
concentrated in vacuo. The residue was purified by column
chromatography (silica gel, eluted with 30−100% EtOAc in hexane)
Biology: Assays for Determination of IC50 Values for
Inhibition of Human OGA and β-Hexosaminidase Activity.
Human OGA was generated from Expi293 cells that had been
transfected with the full-length gene. The enzyme was purified by an
anti-FLAG agarose column and gel filtration. Human β-hexosamini-
dase enzyme from placenta was purchased from Sigma-Aldrich
(Cat#A6152, St. Louis, MO). Enzymatic reactions were carried out in
a reaction containing 50 mM NaH2PO4, 100 mM NaCl and 0.01%
bovine serum albumin (BSA), 0,01% Tween 20, 1 mM dithiothreitol
(DTT, pH 7.4) using 2 mM 4-methylumbelliferyl N-acetyl-β-D-
glucosaminide dihydrate (Sigma-Aldrich, St. Louis, MO, Cat#M2133)
as a substrate. The amount of human OGA or β-hexosaminidase
enzyme used in the reaction was 1 nM or 10 μ units unit/mL. Test
compound of varying concentrations was added to the enzyme prior
to initiation of the reaction. The reaction was performed at room
temperature (RT) in a 384-well plate (Greiner, Frickenhausen,
Germany, Cat#784076) and was initiated by the addition of substrate.
The production of the fluorescent product was measured every 5 min
for 60 min by a SpectraMax M5/M5e Microplate Reader (Molecular
Devices, San Jose, CA) with excitation at 355 nm and emission
detected at 460 nm. Inhibition rate was calculated based on 0%
control wells with enzyme and DMSO, and 100% control wells
without enzyme. Data analysis was done with GraphPad Prism V.5
(GraphPad Software, San Diego, CA) statistical analysis software
using a nonlinear regression for a sigmoidal dose−response with a
variable slope. The IC50 value was defined as the concentration
inhibiting the activity by 50%.
The kinetics experiments of compound 5a or Thiamet-G were
performed with a final concentration of 1 nM human OGA and
various concentrations of 4-methylumbelliferyl N-acetyl-β-D-glucosa-
minide dihydrate obtained by serial 2-fold dilutions starting from 2
mM in an assay buffer (50 mM NaH2PO4, 100 mM NaCl and 0.01%
BSA, 0,01% Tween 20, 1 mM DTT (pH 7.4)). Fluorescence for each
reaction was measured for 60 min using a SpectraMax M5/M5e
Microplate Reader (Molecular Devices, San Jose, CA) with excitation
at 355 nm and emission detected at 460 nm. Vmax and Km values were
calculated using the Michaelis−Menten nonlinear fit in GraphPad
Prism V.5 (GraphPad Software, San Diego, CA).
1
to yield compound 4b (3.48 g, 22%) as colorless crystals. H NMR
(300 MHz, DMSO-d6) δ 2.82 (2H, t, J = 7.5 Hz), 3.47 (2H, d, J = 6.4
Hz), 6.66 (1H, d, J = 5.3 Hz), 7.12−7.34 (5H, m), 7.73 (1H, t, J = 5.7
Hz), 8.23 (1H, brs). MS: [M + H]+ 234.0.
4-(4-Methylpiperidin-1-yl)-N-phenethylpyrimidin-2-amine (5a).
To a mixture of compound 4a (200 mg, 0.94 mmol) and
phenethylamine (0.178 mL, 1.42 mmol) in tert-AmOH (4 mL) was
added DIPEA (0.977 mL, 5.67 mmol) at room temperature. The
mixture was heated at 130 °C for 1 h under microwave irradiation.
The mixture was concentrated in vacuo. The residue was purified by
column chromatography (NH silica gel, eluted with 10−80% EtOAc
1
in hexane) to yield compound 5a (260 mg, 93%) as a yellow oil. H
NMR (300 MHz, DMSO-d6) δ 0.91 (3H, d, J = 6.4 Hz), 0.93−1.11
(2H, m), 1.49−1.71 (3H, m), 2.67−2.88 (4H, m), 3.34−3.48 (2H,
m), 4.30 (2H, d, J = 13.2 Hz), 6.00 (1H, d, J = 6.0 Hz), 6.48 (1H,
brs), 7.14−7.34 (5H, m), 7.76 (1H, d, J = 6.0 Hz). MS: [M + H]+
297.3.
4-Morpholino-N-phenethylpyrimidin-2-amine (5b). Compound
5b was synthesized by general procedure B combinatorially in a 54%
yield. MS: [M + H]+ 285.2.
4-(4-Methylpiperazin-1-yl)-N-phenethylpyrimidin-2-amine (5c).
Compound 5c was synthesized by general procedure B combinato-
rially in a 67% yield. MS: [M + H]+ 298.3.
1-(2-(Phenethylamino)pyrimidin-4-yl)piperidin-4-ol (5d). Com-
pound 5d was synthesized by general procedure B combinatorially in
a 65% yield. MS: [M + H]+ 299.3.
4-(4-Methoxypiperidin-1-yl)-N-phenethylpyrimidin-2-amine
(5e). Compound 5e was synthesized by general procedure B
combinatorially in a 58% yield. MS: [M + H]+ 313.3.
4-(4-Methylpiperidin-1-yl)-N-(2-(pyridin-2-yl)ethyl)pyrimidin-2-
amine (5f). Compound 5f was synthesized by general procedure A
combinatorially in a 62% yield. MS: [M + H]+ 298.3.
4-(4-Methylpiperidin-1-yl)-N-(2-(pyridin-3-yl)ethyl)pyrimidin-2-
amine (5g). Compound 5g was synthesized by general procedure A
combinatorially in a 27% yield. MS: [M + H]+ 298.3.
In-Cell Western (ICW) Assay for Determination of EC50
Values for Cell-Based Inhibition of O-GlcNAcase Activity.
Inhibition of O-GlcNAcase, which removes O-GlcNAc from cellular
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proteins, results in an increase in the level of O-GlcNAcylated protein
in cells. An increase in the amount of O-GlcNAcylated protein was
measured by the ICW assay using an anti-O-GlcNAc antibody RL2
(Abcam, Cambridge, MA) and the Aerius Automated Infrared
Imaging System (LI-COR Biosciences, Lincoln, NE) according to
the manufacturer’s instructions.
Knowledgebase45 (UniProt accession number: O60502). The crystal
structure of BT_4395 OGA in complex with Thiamet-G was
downloaded from the Protein Data Bank46 (PDB ID: 2VVN). This
structure includes the A-chain and B-chain of OGA. The A-chain was
used as a template for the homology model of human OGA. The
homology model of human OGA was built using [Homology
modeling/Prime] in Maestro version 10.4.32 The constructed
homology model was prepared using Protein Preparation Wizard in
Maestro.
Briefly, human neuroblastoma SH-SY5Y cells were plated in 96-
well plates (Corning, Tewksbury, MA, BioCoat Poly-D-Lysine, Cat#
354640) with approximately 1.8 × 104 cells/well. After 48 h
incubation with the compounds at 37 °C, the cells were fixed with
ice-cold methanol for 30 min at RT. Then, the cells were
permeabilized with 0.1% Triton X-100 for 15 min at RT and blocked
with an LI-COR Odyssey Blocking Solution (LI-COR Biosciences,
Lincoln, NE, Cat#927-40000) for 60 min. The cells were stained with
an O-GlcNAc Ab (RL2) Alexa Fluoro 700 (1:500 dilution,
Cat#NB300-524AF700, NOVUS) at RT for 60 min. After three
washes with PBS-T (0.1% Tween/phosphate-buffered saline
(PBS(−))), the integrated fluorescence intensities representing the
protein expression levels were acquired using the software provided
with the imager station. Values were calculated to DMSO-treated cells
as the positive control (100%) and culture medium as the negative
control (0%). The EC50 value was defined as the inflection point of
the curve, the concentration of the test compound increasing the
amount of O-GlcNAcylated proteins by 50%. Emax was defined as the
maximum % of the increased O-GlcNAcylated protein by compounds.
The EC50, 95% confidence intervals, and Emax were analyzed by
triplicate measurements with GraphPad Prism V.5 (GraphPad
Software, San Diego, CA) statistical analysis software using a
nonlinear regression for a sigmoidal dose−response with a variable
slope.
Protein Crystallography. The BT_4395 construct was cloned
into a pFastBac vector containing amino acid residues 22−737
(UniProt: Q89ZI2) with an N-terminal 6XHis tag. The protein was
expressed in baculovirus-infected B. thetaiotamicron cells. Purification
was with Ni-affinity, gel filtration, and 2006 NSMB concentration
steps followed by cleavage step (TEV) followed by NiRev and SEC.
200. Protein buffer was 20 mM 4-(2-hydroxyethyl)-1-piperazineetha-
nesulfonic acid (HEPES) pH 7.5, 300 mM NaCl at a final
concentration of 11 mg/mL. Hanging drop crystallization using a
matrix around the PDB 2CHN condition: 0.5 M NaAcetate, 15%
PEG 3350, 0.1 M MES pH 6.0, 20% glycerol. The final conditions of
the crystal chosen for complexing were 15−19% PEG 3350, 10%
glycerol, 0.34−0.4 M NaAcetate, 0.1 M MES 6.0. The crystal was
soaked for 60 h with compound at a concentration of 5 mM. X-ray
diffraction was collected at Advanced Light Source beamline 5.0.3
under cryo-temperature. Diffraction images were processed with
HKL2000 and solved and refined with CCP4 tools Phaser and
Refmac, respectively.43 Model was built into the electron density
using Coot.44
Virtual Screening. An in-house compound library comprising a
few million compounds was screened by virtual screening. The
compounds were prepared using LigPrep to generate all possible
isomers and ionization states and build one 3D structure per isomer/
ionization state. For ligand-based virtual screening, multiple
conformations were generated using OMEGA version 2.5.47 with
the following parameter sets: RMS = 0.3 and number of
conformations = 20 000. As templates for the ligand-based virtual
screening, the crystal structures of complexes between various
bacterial OGA and their inhibitory ligands were downloaded from
the Protein Data Bank (PDB IDs: 2VVN, 2VVS,14 2XJ7,15 5ABG,16
2WB5,17 and 2WCA18) and the coordinates of the ligands were
extracted. Shape-based similarity search of in-house compounds was
carried out against reported compounds using ROCS version 3.234
and then the top 100 000 molecules, in terms of their
TanimotoCombo scores, were extracted per one query compound.
After removing duplicate molecules, the docking of the remaining
compounds was performed into the constructed homology model of
human OGA employing the Glide SP mode.32 Compounds forming
hydrogen bonds with at least one residue among Gly67, Asp174, and
Asp285 were selected by the Pose Filter function in Maestro.
Thereafter, the selected compounds were filtered on the basis of their
physicochemical properties (molecular weight ≤ 350, A log P ≤ 5,
number of hydrogen bond donors ≤ 4, number of hydrogen bond
acceptors ≤ 8, number of rotatable bonds ≤ 8, and topological polar
surface area ≤ 125) and via PAINS queries using the Pipeline Pilot
version 17.2.40 The remaining compounds were divided into 2681
clusters employing the fingerprint-based clustering method using
ECFP_6 with Pipeline Pilot.
Docking Model. Compounds were prepared using LigPrep in
Maestro and docked into the homology model using the Glide SP
mode. The top-ranked binding pose was refined by energy
minimization including the flexible amino acid residues within 6 Å
of the compound.
WaterMap Analysis. WaterMap32,37,38 was run for the docking
model of compound 2 with the hOGA homology model, where the
compound was removed in the MD simulation.
Molecular Dynamics (MD) Simulation. Desmond32 was used
for 50 ns MD simulation. The docking model of compound 5i with
the human OGA homology model was used as the initial structure of
the MD simulation. During simulation, the temperature and the
pressure were maintained at 300 K and 1.01 bar, respectively.
Brain Distribution in Mice. Compound 5i was suspended in
0.5% (w/v) methylcellulose solution and orally administered to
C57Bl/6J mice (n = 3). At 0.5 and 1 h after oral administration (30
mg/kg), blood and brain samples were collected. The blood samples
were centrifuged to obtain the plasma fraction. The brain samples
were homogenized in saline to obtain the brain homogenate. The
plasma and brain homogenate samples were deproteinized with
acetonitrile containing an internal standard. After centrifugation, the
supernatant was diluted with 0.2% (v/v) formic acid in 10 mmol/L
ammonium formate (pH 3). The compound concentrations were
measured by LC/MS/MS. After determination of concentration in
plasma (Cplasma) and brain (Cbrain), Kp,uu was calculated by the
equation as follows
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01712.
Molecular formula strings (CSV)
Dose−response curve of 2 for hOGA inhibition (Figure
S1); surface map characterized by SiteMap (Figure S2);
kinetic assay to determine Km for substrate-competition
of 5a (Figure S3); kinome map of tested kinase for 5i
(Figure S4); MD calculation for the docking model of 5i
in complex with hOGA (Figure S5); data collection and
refinement statistics of crystallography (Table S1); and
docking model of 2 in complex with human OGA
(PDB) (PDF)
Kp,uu = (fu,brain × Cbrain)/(fu,plasma × Cplasma
)
where fu,brain and fu,plasma represent the unbound fraction in the
plasma and brain, respectively. The fu,brain and fu,plasma were
determined by an equilibrium dialysis.
Computational Chemistry: Homology Modeling. The amino
acid sequence of human OGA was collected from UniProt
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Journal of Medicinal Chemistry
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Article
Accession Codes
performance liquid chromatography; LLE, ligand lipophilicity
efficiency; MeCN, acetonitrile; OGA, O-GlcNAcase; O-
GlcNAc, O-linked N-acetylglucosamine; OGT, O-GlcNAc
transferase; PDB, Protein Data Bank; SAR, structure−activity
relationship; tert-AmOH, 2-methyl-2-butanol; TFA, trifluoro-
acetic acid; THF, tetrahydrofuran
Atomic coordinates and structure factors for the crystal
structure of BT_4395 OGA in complex with compound 5a
have been deposited with the RCSB Protein Data Bank (PDB
code 7K41).
AUTHOR INFORMATION
Corresponding Author
Michiko Tawada − Research, Takeda Pharmaceutical
Company Limited, Fujisawa, Kanagawa 251-8555, Japan;
orcid.org/0000-0002-1349-0059;
■
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Authors
Makoto Fushimi − Research, Takeda Pharmaceutical
Company Limited, Fujisawa, Kanagawa 251-8555, Japan
Kei Masuda − Research, Takeda Pharmaceutical Company
Limited, Fujisawa, Kanagawa 251-8555, Japan
Huikai Sun − Takeda California, Inc., San Diego, California
92121, United States; orcid.org/0000-0002-4593-1306
Noriko Uchiyama − Research, Takeda Pharmaceutical
Company Limited, Fujisawa, Kanagawa 251-8555, Japan
Yohei Kosugi − Research, Takeda Pharmaceutical Company
Limited, Fujisawa, Kanagawa 251-8555, Japan;
orcid.org/0000-0001-9318-4723
Weston Lane − Takeda California, Inc., San Diego, California
92121, United States
Richard Tjhen − Takeda California, Inc., San Diego,
California 92121, United States
Satoshi Endo − Research, Takeda Pharmaceutical Company
Limited, Fujisawa, Kanagawa 251-8555, Japan
Tatsuki Koike − Research, Takeda Pharmaceutical Company
Limited, Fujisawa, Kanagawa 251-8555, Japan;
orcid.org/0000-0002-3264-1406
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01712
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
We acknowledge Koichi Iwanaga, Atsuko Ochida, and Izumi
Nomura for the excellent technical assistance with conducting
combinatorial chemistry; Takanobu Kuroita for valuable
suggestions to drug design; Terufumi Takagi for helpful
suggestions to computational chemistry; Yoshihiko Hirozane
for conducting kinase panel screening; Shoichi Okubo and
Takashi Ito for preparing the human OGA cDNA and full-
length proteins; and Koji Murakami, Shunya Suzuki, and
biology group members for the valuable scientific discussion.
We thank the staff of the Berkeley Center for Structural
Biology, which operates Advanced Light Source beamline
5.0.3. The Berkeley Center for Structural Biology is supported
in part by the National Institutes of Health and National
Institute of General Medical Sciences. The Advanced Light
Source is supported by the Director, Office of Science, Office
of Basic Energy Sciences of the U.S. Department of Energy
under contract number DEAC02-05CH11231.
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ABBREVIATIONS USED
■
CNS, central nervous system; CNS-MPO, central nervous
system-multiparameter optimization; dba, DIPEA, N,N-diiso-
propylethylamine; DMSO, dimethyl sulfoxide; HPLC, high-
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Zandberg, W. F.; Shan, X.; Varghese, V.; Chan, S.; Davies, G. J.;
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