Multistage screening reveals chameleon ligands of the human farnesyl pyrophosphate synthase: Implications to drug discovery for neurodegenerative diseases

Article abstract of DOI:10.1021/jm500629e

Human farnesyl pyrophosphate synthase (hFPPS) is the gate-keeper of mammalian isoprenoids and the key target of bisphosphonate drugs. Bisphosphonates suffer from poor "drug-like" properties and are mainly effective in treating skeletal diseases. Recent investigations have implicated hFPPS in various nonskeletal diseases, including Alzheimer's disease (AD). Analysis of single nucleotide polymorphisms in the hFPPS gene and mRNA levels in autopsy-confirmed AD subjects was undertaken, and a genetic link between hFPPS and phosphorylated tau (P-Tau) levels in the human brain was identified. Elevated P-Tau levels are strongly implicated in AD progression. The development of nonbisphosphonate inhibitors can provide molecular tools for validating hFPPS as a therapeutic target for tauopathy-associated neurodegeneration. A multistage screening protocol led to the identification of a new monophosphonate chemotype that bind in an allosteric pocket of hFPPS. Optimization of these compounds could lead to human therapeutics that block tau metabolism and arrest the progression of neurodegeneration.

Full text of DOI:10.1021/jm500629e

Article
pubs.acs.org/jmc
Multistage Screening Reveals Chameleon Ligands of the Human
Farnesyl Pyrophosphate Synthase: Implications to Drug Discovery
for Neurodegenerative Diseases
Joris W. De Schutter,†,# Jaeok Park,‡,# Chun Yuen Leung,† Patrick Gormley,§ Yih-Shyan Lin,†
Zheping Hu,†,∇ Albert M. Berghuis,‡,∥,⊥ Judes Poirier,§ and Youla S. Tsantrizos*,†,‡,⊥
†Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, Quebec H3A 0B8, Canada
‡Department of Biochemistry, McGill University, 3655 Promenade Sir William Osler, Montreal, Quebec H3G 1Y6, Canada
§Douglas Mental Health University Institute, 6825 Lasalle, Verdun, Quebec, H4H 1R3, Canada
∥Department of Microbiology and Immunology, McGill University, 3775 Rue University, Montreal, Quebec H3A 2B4, Canada
⊥Groupe de Recherche Axe
H3G 0B1, Canada
́ ́
sur la Structure des Proteines, McGill University, 3649 Promenade Sir William Osler, Montreal, Quebec
S
* Supporting Information
ABSTRACT: Human farnesyl pyrophosphate synthase (hFPPS)
is the gate-keeper of mammalian isoprenoids and the key target of
bisphosphonate drugs. Bisphosphonates suffer from poor “drug-
like” properties and are mainly effective in treating skeletal
diseases. Recent investigations have implicated hFPPS in various
nonskeletal diseases, including Alzheimer’s disease (AD). Analysis
of single nucleotide polymorphisms in the hFPPS gene and mRNA
levels in autopsy-confirmed AD subjects was undertaken, and a
genetic link between hFPPS and phosphorylated tau (P-Tau)
levels in the human brain was identified. Elevated P-Tau levels are
strongly implicated in AD progression. The development of
nonbisphosphonate inhibitors can provide molecular tools for
validating hFPPS as a therapeutic target for tauopathy-associated
neurodegeneration. A multistage screening protocol led to the identification of a new monophosphonate chemotype that bind in
an allosteric pocket of hFPPS. Optimization of these compounds could lead to human therapeutics that block tau metabolism
and arrest the progression of neurodegeneration.
closing of the active site cavity, sequestering its substrates from
bulk water.1a,c
INTRODUCTION
■
Human farnesyl pyrophosphate synthase (hFPPS) catalyzes the
elongation of dimethylallyl pyrophosphate (DMAPP) to
geranyl pyrophosphate (GPP) and then to farnesyl pyrophos-
phate (FPP) via the sequential condensation of DMAPP and
then GPP with an isopentenyl pyrophosphate (IPP) unit. The
active site cavity of this enzyme is characterized by two highly
charged binding subpockets, making the identification of active
site inhibitors, with physicochemical properties appropriate for
treating nonskeletal diseases, extremely challenging. The allylic
subpocket (DMAPP/GPP binding site) is composed of two
aspartate-rich motifs that bind the pyrophosphate moiety of the
substrate via metal mediated interactions.1 The other subpocket
is lined with basic residues that engage in direct salt-bridge
interactions (K57, R60, R113) or water-mediated interactions
(R112, R351) with the pyrophosphate moiety of IPP (Figure
1a).1 The initial occupancy of the allylic subpocket induces a
protein conformational change from the “open” to a “partially-
closed” state, which fully defines the shape of the IPP binding
site. The subsequent binding of IPP leads to the complete
Currently, nitrogen-containing bisphosphonates (N-BPs) are
the only clinically useful inhibitors of hFPPS; the best examples
from this class of drugs include zoledronic acid (1) and
risedronic acid (2a). These drugs bind exclusively to the allylic
subpocket and serve as chemically stable pyrophosphate mimics
of DMAPP/GPP (Figure 1a).1 Because of their highly charged
nature and affinity for bone, N-BPs exhibit poor cell-membrane
permeability, rapid clearance from the systemic circulation, and
almost negligible distribution to nonskeletal tissues.2 Con-
sequently, these drugs are mainly used in treating skeletal
disorders such as osteoporosis and tumor-induced osteolytic
metastases.3 However, hFPPS is the gatekeeper for the
prenylation and activation of many small GTPases that play
an essential role in a plethora of cellular functions including cell
signaling,4 proliferation, and synaptic plasticity.5 Inhibition of
Received: April 22, 2014
© XXXX American Chemical Society
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Figure 1. Binding pockets of hFPPS. (a) Structure of the hFPPS/5m/
IPP complex (PDB ID: 4H5E). (b) Structure of the hFPPS/4/Pi
complex (PDB ID: 3N6K). The protein surface within 4 Å distance of
the bound ligands is rendered semitransparent to indicate the binding
pockets. The carbon backbone of IPP, 5m, and 4 are highlighted in
cyan, green, and magenta, respectively. Oxygen, nitrogen, and
phosphorus atoms are colored in red, blue, and orange, respectively.
Yellow and red spheres represent the Mg2+ ions and water molecules,
respectively.
hFPPS leads to reduction of cell viability and has been
implicated in the antitumor clinical benefits of N-BPs.3,6
Inhibition of hFPPS also induces activation of γδ T cells,
providing immunosurveillance against tumors.7 Additionally,
overexpression of hFPPS and high levels of farnesyl
pyrophosphate (FPP) in the brain tissue are believed to play
a key role in neurodegeneration;8 the latter is also linked to the
metabolism of phosphorylated tau (P-Tau) protein in the
human brain. It is indeed possible that the prenylation cascade
from FPP → GGPP → RhoA-cdc42 →GSK3-β → P-Tau is
largely (or in part) responsible for Alzheimer’s-associated tau
phosphorylation and tangle formation of neurons. Elevated
levels of P-Tau lead to neurofibrillary tangle formation in the
brain, a pathological hallmark of neurodegeneration and the
Alzheimer’s disease (AD). In this study, we report a genetic
relationship between hFPPS and P-Tau concentration in the
cortical area of the human brain in AD subjects.
The potential value of hFPPS as a therapeutic target (beyond
for bone-related diseases) has fuelled efforts toward the
identification of nonbisphosphonate inhibitors for this target.
Fragment-based screening by NMR and X-ray crystallography
led to the first discovery of compounds (e.g. 3) that could bind
in an allosteric pocket of hFPPS near the IPP subpocket.9
Subsequent optimization of these hits provided nonbi-
sphosphonate allosteric inhibitors with nanomolar potency
(e.g., 4; Figure 1b). More recently, in silico studies identified
bisamidine-type inhibitors with micromolar potency;10 how-
ever, the binding of these molecules in the allosteric pocket has
not been confirmed. In this report, we describe the discovery of
thienopyrimidine bisphosphonates (ThP-BPs; 6) that exhibit a
mixed binding mode and can interact with both the allylic
subpocket of the active site and the allosteric pocket of hFPPS.
Structural remodeling of these compounds to the mono-
phosphonate derivatives (ThP-MP; 7) resulted in a new
chemotype of hFPPS inhibitors that bind selectively in the
allosteric pocket and exhibit equivalent in vitro potency to
compound 4. In a preliminary biological evaluation, we
examined the ability of several, structurally diverse hFPPS
inhibitors to modulate intracellular P-Tau levels in human
immortalized neurons. Our combined medicinal chemistry
efforts and genetic analysis data clearly supports a biochemical
link between prenylation and the accumulation of P-Tau in
neurons and the AD brain that can potentially be alleviated by
inhibiting hFPPS.
RESULTS AND DISCUSSION
Screening for Allosteric Inhibitors of hFPPS. The
■
bisphosphonate moiety is a chemically stable bioisostere of
pyrophosphate. Mindful of the direct interactions that the
pyrophosphate moiety of IPP makes with the positively charged
residues in its binding pocket (K57, R60, and R113; Figure
1a,b) and the proximity of these residues to the allosteric
pocket, we speculated that in the absence of Mg2+ ions
bisphosphonates may also bind near (or in) the allosteric
pocket. The molecular recognition elements surrounding this
pocket could conceivably have practical bearings in allowing
pyrophosphate-containing metabolites to discriminate between
the allylic subpocket and the R/K-rich IPP/allosteric site on the
basis of the protein conformation. It is noteworthy that
although the biological role of this allosteric pocket is still
unknown, on the basis of its amphiphilic nature and proximity
to the active site, a regulatory role in feedback inhibition by
downstream isoprenoids is plausible and has been proposed.9
Furthermore, a mixed binding mode of pyrophosphate-
containing isoprenoids is not unprecedented; binding of IPP
to the allylic subpocket of hFPPS1b and of DMAPP in the IPP
subpocket of FPPS from Trypanosoma cruzi (PDB ID: 1YHL)11
have been previously reported.
In earlier studies, we reported the synthesis and in vitro
activity of aminopyridine-based bisphosphonate (N-BP; 5) and
thienopyrimidine-based bisphosphonate (ThP-BP; 6) inhibitors
of hFPPS (Table 1).12 In this study, we investigated their
binding to hFPPS using differential scanning fluorimetry
(DSF). The thermal unfolding of hFPPS (ΔTm) in the
presence and absence of these ligands was compared to their
in vitro potency (Figures 2 and 3). Initial validation of our DSF
assay was carried out with a number of control experiments
using known compounds. For example, the DSF data of the
hFPPS/2a and hFPPS/2a/IPP complexes (ΔTm of 22 0.5
and 30 0.5 °C, respectively) were found to be consistent with
literature DSC data.1a Additionally, the thermal stability of the
hFPPS/4 complex (ΔTm = 8 0.5 °C) was found to be lower
than that of hFPPS/2a and invariable by the presence or
absence of IPP and Mg2+ ions. These observations are in
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Table 1. Library of hFPPS Inhibitors Used for Screening: N-BPs and ThP-BPs with General Structure 5 and 6, Respectively
(The Synthesis of These Compounds Were Previously Reported12)
agreement with the lower intrinsic potency of 4 (compared to
2a) and its competitive binding with IPP.9
thermal stability that was 3−4 °C higher than the unliganded
enzyme. In general, the hFPPS/6 complexes exhibited higher
thermal stability than the hFPPS/5 complexes in both the
presence and absence of Mg2+ ions (albeit fewer ThP-BPs were
tested). Under Mg2+-free conditions, several hFPPS/6 com-
plexes exhibited thermal stability equivalent to that of the
hFPPS/4 complex (Figure 3b).
To further characterize the binding of our compounds in the
presence and absence of Mg2+ ions, competitive binding
between pairs of ligands was investigated using 1H line
broadening NMR. Broadening of the 1H resonances of inhibitor
4 was observed in the presence of Mg2+ ions and hFPPS
(Supporting Information Figure 1), suggesting fast exchange
between the free and enzyme-bound state of 4. Addition of 2a
A number of hFPPS/5 and hFPPS/6 complexes were then
evaluated by DSF in the presence and absence of the enzyme’s
Mg2+ cofactor (Table 1; Figure 3). To minimize the impact of
false positive hits due to nonselective binding, only inhibitors
with IC50 values below 2 μM and selectivity >100-fold for
inhibiting hFPPS vs hGGPPS were included in this screen
(Figure 2); N-BPs are known to inhibit related enzymes, such
as hGGPPS13 and hSQS.14 In the presence of Mg2+ ions, a
good correlation between the ΔTm and IC50 values for the N-
BPs (5) was observed (Figure 3a). Predictably, in the absence
of Mg2+ ions binding was virtually abolished (ΔTm <2 °C 0.5
°C). Exceptionally, few complexes (e.g. hFPPS/5n), exhibited
C
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Figure 4. 1H line broadening NMR studies exploring competitive
binding of inhibitors 4 and 5n in the absence of Mg2+ ions. Stacked
spectra of compound 5n free in solution (D2O and buffer) and in the
presence of hFPPS and 4; for clarity, only the aromatic resonances are
shown, the molar ratio of ligands and protein are indicated next to
each spectra and the proton resonances of 5n are indicated at the
bottom of the stacked spectra (in blue).
Figure 2. Screening protocol (SI, selectivity index = potency ratio in
hFPPS/hGGPPS).
to the same NMR sample did not restore the 1H resonances of
4 (Supporting Informationm Figure 1), indicating that 2a and 4
do not compete for binding to hFPPS. This is in complete
agreement with the structure of the hFPPS/1/3 ternary
complex (PDB ID: 3N46), which demonstrated that the
allosteric pocket and the allylic subpocket can be simulta-
neously occupied.9 In contrast, competitive binding was
detected between 4 and IPP (Supporting Information Figure
2), consistent with the expected electrostatic/steric clash
between one carboxylic acid moiety of 4 and the pyrophosphate
of IPP (PDB ID: 3N6K).9 In the absence of Mg2+ ions, our
NMR data clearly showed competitive binding between
inhibitors 5n and 4 (Figure 4). However, in the presence of
Mg2+ ions, the data was inconclusive and we could not rule out
that 5n was competing for binding with both 2a and 4; similar
observations were made with analogue 6a.
site and the allosteric pocket (Figure 5). ThP-BP 6a is the first
chemotype to provide conclusive evidence that bisphospho-
nates can indeed bind in the allosteric pocket of hFPPS, at least
in the absence of the metal cofactor. From here on, we will refer
to hFPPS/6 complexes as the hFPPS/6(act) and hFPPS/6(allo)
complexes to indicate binding of the ligand in the active site
and allosteric pocket, respectively (part a vs part b of Figure 5;
PDB IDs 4JVJ vs 4LPG, respectively).
We previously reported the co-crystal structure of the
hFPPS/6a(act) complex (PDB ID 4JVJ; Figure 5a).12d In the
hFPPS/6a(allo) complex, the p-tolyl substituent is buried in the
allosteric pocket, engaging in π-stacking interactions with the
side chains of N59 and F206 (Figure 5b). The alkyl portions of
the L344 and K347 side chains are within van der Waals radius
and provide additional hydrophobic interactions. Edge-to-face
π-stacking interactions are also observed between the
thienopyrimidine core and F239, with the pyrimidine ring
partially solvent exposed (Figure 5b). A noteworthy difference
in the allosteric bindings of 6a and 4 is that 4 engages in direct
Co-crystallization of 6a bound to hFPPS in both the
presence and absence of Mg2+ ions provided proof that this
compound can bind to both the allylic subpocket of the active
Figure 3. Correlation of in vitro potency in inhibiting hFPPS to the thermal stability of the hFPPS/inhibitor complex in the presence and absence of
Mg2+ ions. DSF assays were run with 4 mM hFPPS protein and 40 mM inhibitor. (a) IC50 vs ΔTm of hFPPS/N-BP complexes. (b) IC50 vs DTm of
hFPPS/ThP-BP and hFPPS/4 complexes.
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Figure 5. Co-crystal structures of hFPPS/ligand complexes. (a) Complex hFPPS/6a(act)/Pi (PDB ID: 4JVJ); metal-coordinated water molecules and
some other interactions between the protein and the inhibitor are omitted for clarity. (b) Complex hFPPS/6a(allo)/Pi (PDB ID: 4LPG). (c) ITC data
for the binding of compounds 6a and 7 in the absence of Mg2+ ions. (d) Structure of the hFPPS/7/Pi complex (PDB ID: 4LPH).
interactions with the side chains of K57 and R60 via one of its
carboxylic acids, whereas the bisphosphonate of 6a does not
form any direct interaction but only water-mediated H-bonds
with R60 and D243; the latter residue (D243) is typically
involved in metal-mediated interaction with bisphosphonates
that are bound in the allylic subpocket (part a vs part b of
Figure 5). Surprisingly, the bisphosphonate of 6a is bound very
close to an inorganic phosphate ion (Pi) occupying the IPP
subpocket (a distance of 2.64 Å between OAX and O3; Figure
5b). Although such proximity between two negatively charged
ions is puzzling, the identity and location of both was
unambiguously confirmed by anomalous scattering data
(Supporting Information Figure 3). Presumably, the negative
charge of the Pi is neutralized by the dipole of the αC helix and/
or R60 (a distance of 3.15 Å was observed between a
guanidinium nitrogen and a phosphate oxygen).
The relative contributions of the two binding modes of 6a
under biologically relevant conditions are difficult to decipher.
Whether they both contribute to the intrinsic potency of 6a
(IC50 = 22 nM) is equally unclear; in vitro evaluation of hFPPS
activity requires Mg2+ as the cofactor, which biases the binding
of bisphosphonates in favor of the allylic subpocket. Nonethe-
less, under Mg2+-free conditions, ITC studies suggested a Kd of
∼56 μM for the binding of 6a, presumably in the allosteric
pocket only (Figure 5c). The Kd of the corresponding mono-
phosphonate analogue 7 was ∼9 μM and in close agreement
with its IC50 value of 4.5 μM (Table 2). More importantly, in
the presence of Mg2+ ions, competitive binding of 7 was
unambiguously observed with compound 4 but not with 2a.
These results suggest that under biologically relevant conditions
inhibitor 7 binds selectively in the allosteric pocket (Figure 6).
It is noteworthy that previous attempts to remove one of the
phosphonate moieties of N-BPs led to complete loss of potency
(e.g., 2b vs 2c; Table 2).15
Table 2. Inhibition Data for Key Compounds
a
e
f
compd
hFPPS IC50 (μM)
P-Tau/T-Tau ratio
LDH (%)
control
1
0.012
0.006
0.004
nd
0
0.003
0.006
+60
+60
nd
nd
+3
0%
nd
−1
nd
nd
nd
nd
nd
−7
2a
2b
2c
4
b
0.033
b
>800
nd
c
0.92
0.004
0.007
nd
5n
6a
6f
0.018
0.022
0.014
4.5
0.009
nd
7
d
8
>20
nd
9
26
nd
d
10
11
12
>20
nd
3.3
1.2
nd
0.010
a
The potencies of all analogues of general structure 5 and 6 in
inhibiting hFPPS were previously reported.12 IC50 reported by
b
Dunford et al.15 IC50 of 0.2 μM was reported by Jahnke et al. using a
c
different assay protocol.9 Inhibition of 40% and 27% was observed for
d
e
8 and 10, respectively, at 20 μM. P-Tau/T-Tau ratios in human
neuroblastoma SH-SY5Y cells were determined at a fixed concen-
tration of 100 nM inhibitor after an incubation period of 24 h; average
value of two assays; deviation of <5% was observed. Average %
activity of lactate dehydrogenase observed (LDH) compared to the
untreated control (set arbitrarily to zero LDH activity); assays run in
triplicate and a standard deviation of ∼10% was observed.
f
We subsequently confirmed the binding of 7 in the allosteric
pocket by X-ray crystallography (Figure 5d). The overall
structures of the hFPPS/7 and hFPPS/6a(allo) complexes are
very similar, with the core (Cα) rmsd value of 0.27 Å.
Alignment of the two structures showed clear superposition of
E
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Figure 6. Side-by-side comparison of 1H line broadening NMR studies exploring competitive binding of hFPPS inhibitor 7 with either 4 or 2a in the
presence of Mg2+ ions. Buffer: BSA 20 mg/mL, Tris 50 mM, pH 7.7, MgCl2 2 mM, TCEP 0.5 mM. For clarity, only select regions of the spectra are
shown; the molar ratio of ligands and protein are indicated next to each spectra. Stacked spectra on the left are those of compound 7 free in solution
and in the presence of hFPPS and compound 4; the data clearly shows competitive binding even in the presence of Mg2+ ions. Stacked spectra on the
right are those of compound 7 free in solution and in the presence of hFPPS and compound 2a, clearly showing that binding of the two compounds
is not competitive in the presence of Mg2+ ions. All proton resonances are labeled at the top of the spectra in red, blue, and black for compounds 4, 7,
and 2a, respectively.
the thienopyrimidine cores, the tolyl side chains and the Pi
(part b vs part d of Figure 5). Although the phosphonate
moiety of 7 did not overlap with either of the two
phosphonates of 6a, it maintained a water-mediated interaction
with R60. In an attempt to gain more insight as to the
importance of this phosphonate moiety, and possibly allow this
moiety to form a direct salt-bridge with R60, we synthesized
analogue 8, having a longer linker between the phosphonate
and the thienopyrimidine core. A significant drop in potency
was observed (Table 2), likely due to the increased conforma-
tional freedom and entropic penalty associated with the binding
of 8. We also investigated the impact of decreasing the acidity
of this pharmacophore by replacing the exocyclic NH with a
methylene linker at C-4 (pKa of P(O)O−OH of ∼6.8 and ∼7.7
for analogues 7 and 9, respectively) and by completely
replacing the phosphonate with a carboxylic acid moiety (i.e.,
10). A correlation between loss in potency and decrease in the
acidity of this moiety was clearly observed, confirming the
importance of this pharmacophore (Table 2). Finally, minor
substitutions on the tolyl side chain (e.g., 11; IC50 = 3.3 μM)
and the Cα to the phosphonate were well tolerated and appear
to be additive in improving the potency of this class of
compounds. In the present study, analogue 12 (IC50 = 1.2 μM)
was the most potent hit identified, exhibiting equivalent
potency to compound 4 in inhibiting hFPPS in our in vitro
assay (IC50 = 0.92 μM). More extensive SAR investigations and
further optimizing of these allosteric inhibitors is currently in
progress.
Treatment of 13 first with Br2 in acetic acid, followed by
POCl3, provided the 4-chloro-6-bromo intermediate 14 in
moderate overall yield (∼40% over the two steps). Analogues 7,
11, and 12 were prepared after sequential SNAr displacement of
the C-4 chloro with an aminodiethylphosphonate (prepared as
previously reported17), followed by Suzuki cross-coupling with
an aryl boronate and final deprotection of the phosphonate
esters with TMSBr/MeOH (Scheme 1). Alternatively, the
Suzuki reaction could be carried first to obtain the key building
block 4-chloro-6-(p-tolyl)thieno[2,3-d]pyrimidine, followed by
the SNAr displacement of the C-4 chloro with diethyl (2-
aminoethyl)phosphonate to give the diethyl ester of analogue 8,
or the unprotected glycine to obtain directly compound 10,
with similar overall yields. These two alternative approaches
provide easy access to library synthesis of analogues with
structural diversity at both the C-4 and C-6 of the
thienopyrimidine scaffold.
Suzuki−Miyaura cross coupling of trifluoro(vinyl) borane
with the 4-chlorothienopyrimidine, followed by modified
Lemieux−Johnson oxidation,18 gave the aldehyde 15 in good
overall yields (Scheme 1). Horner−Wadsworth−Emmons type
condensation of intermediate 15 with the sodium anion of
tetraethyl methylenediphosphonate gave the α,β-unsaturated
monophosphonate diethyl ester.19 Attempts to remove the
ethyl esters of this intermediate (under the usual condition of
TMSBr/MeOH) led to some decomposition, therefore, this
intermediate was first hydrogenated before the deprotection
step to give compound 9 (Scheme 1).
Synthesis of Ligands Binding to the Allosteric Pocket.
The synthesis of the new compounds 7−12 was initiated from
the key intermediate 13, prepared as previously reported.16
Relevance of hFPPS in Modulating Phospho-Tau
Levels in the Human Brain. Elevated plasma cholesterol
levels are known among the vascular risk factors of the
F
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a
Phospho-tau production in the brain is regulated by a select
number of isoprenoid-activated small G proteins that control
the function of the GSK3-β kinase. GSK3-β is believed to be
responsible for high levels of tau phosphorylation in neurons.25
This hypothesis prompted us to carefully examine the
relationship between the hFPPS gene and tau metabolism in
the Alzheimer’s disease (AD).26 We examined a cohort of 751
pairs of AD and age-matched control subjects to map
approximately 535000 polymorphisms in each of the AD
cases and control subjects. Five single nucleotide poly-
morphisms (SNPs) were examined in the hFPPS gene loci
(Supporting Information Figure 4). Frontal cortex brain
samples (n = 116) from autopsy-confirmed cases of Alzheimer’s
disease and age-matched control subjects were prepared
according to an established protocol27 prior to ELISA
quantification of cortical total tau (T-Tau) and phosphorylated
tau (P-Tau) protein levels. While none of the markers were
found to exhibit significant association with sporadic AD, a
specific and dose dependent genetic association was found
between variant rs4971072 (SNP:A/G) in the promoter region
of the hFPPS gene and P-Tau protein levels (p < 0.02) in the
cortical area of the AD brain (Figure 7a). We also examined a
subset of AD (n = 34) and age-matched control brains (n = 24)
for the mRNA prevalence of hFPPS using QrtPCR as
previously reported.28 Human FPPS mRNA levels were
markedly elevated in the cortical cortices of the AD versus
the control subjects (Figure 7b), consistent with a hyper-
activation of the isoprenoid pathway in Alzheimer’s disease.
Although these finding do not imply that hFPPS is causally
related to Alzheimer’s disease, they support a genetic
association between specific genetic variants in hFPPS
(rs4971072) and the P-Tau concentrations in the human AD
brain. One of the early pathological features of common
Alzheimer’s disease is the progressive accumulation of ribbon-
like intraneuronal tangles made of polymer of the phosphory-
lated tau protein (P-Tau), a key cytoskeleton structural
component of neurons. Human FPPS clearly affects the
accumulation of the pro-toxic P-Tau and may accelerate the
progression of AD pathology.26
Scheme 1. Synthesis of Allosteric Inhibitors of hFPPS
a
Conditions: (a) Br2, AcOH, 80 °C (63%); (b) POCl3, 95 °C (58%);
(c) H2NCHR1PO(OR′)2 (16), Et3N, dioxane, 100 °C, 18−24 h (40−
60%); (d) Pd(PPh3)4, K2CO3, dioxane/H2O (1:1), 100 °C, 1 h (60−
70%); (e) TMSBr, MeOH, RT, 72 h (>98% conversion, 50−80%
isolated yield); (f) potassium trifluoro(vinyl)borate, PdCl2(dppf)-
CH2Cl2, Et3N, i-PrOH/H2O, 100 °C, 1 h (72%); (g) (i) OsO4, NMO,
2,6-lutidine, acetone/H2O, RT, 2 h, (ii) NaIO4, RT, 1 h (64%); (h) (i)
tetraethyl methylenediphosphonate, NaH, THF, 0 °C to RT, 1 h, (ii)
H2, Pd/C, RT, 1 h, then step e (45% isolated yield for the 3 steps); (i)
H2NCH2CO2H, Na2CO3, dioxane/H2O (1:1), 100°C, 3 h (60%).
Alzheimer’s disease (AD).20,21 Although not a universal finding,
treatment of middle-aged individuals for hypercholesterolemia
with statins has been shown to confer some level of
neuroprotection against late-life development of AD.22 Addi-
tionally, statin treatment has been shown to reduce the
cerebrospinal fluid phospho-tau content in the human brain.23
These finding are consistent with the quasi-absence of cortical
neurofibrillary tangles in autopsy-confirmed cognitively intact
subjects who had used statins for several years as opposed to
nonusers.24
These preliminary results prompted us to also examine the
ability of hFPPS inhibitors to block tau metabolism in human
neuroblastoma SH-SY5Y cells. Cells were treated with both
bisphosphonate and nonbisphophonate inhibitors at concen-
trations of 100 and 500 nM for a period of 24 h before they
were lysed and their T-Tau and P-Tau levels were quantified
using an established ELISA test.27 Toxicity to the neurons was
Figure 7. Genetic association between hFPPS and P-Tau metabolism. (a) Effects of rs4971072 variant copy number on P-Tau concentrations in
cortical area of the AD brain. (b) Human cortical FPPS mRNA prevalence in the cortical area in AD brain vs control. AD mean Braak stage = 4.9
1.8; age of AD onset = 71 10; age of death = 74 12 and 78 9 for control and AD, respectively.
G
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estimated using a routine lactate dehydrogenase (LDH) assay
and found to increase sharply after 48 h of incubation with the
inhibitors tested. Similarly, some toxicity was observed with
most inhibitors if tested at concentrations of 500 nM or higher,
prohibiting a clear determination of P-Tau/T-Tau levels.
Although a significant decrease (≥50% decrease) in the P-
Tau/T-Tau levels was observed with the most potent N-BP
inhibitors 1 and 2a, significant toxicity was also observed
(∼60% higher LDH activity as compared to the untreated
control) even at concentrations of 100 nM (Table 2). In
contrast, the bisphosphonate inhibitors 5n and 6f and
nonbisphosphonate inhibitor 4 induced a significant decrease
in P-Tau/T-Tau ratio without significant toxicity, whereas the
monophosphonate inhibitor 12 exhibited only minor effects
(Table 2).
neurotoxic neurofibrillary tangles, an established hallmark of
AD brain pathology. Preliminary evaluation of hFPPS inhibitors
in neuroblastoma cells suggested that hFPPS can modulate tau
metabolism; confirmation of these results is pending the
identification of more potent/less toxic inhibitors of hFPPS
with good cell-membrane permeability. Nonetheless, our
collective data supports further investigation of hFPPS as a
potential therapeutic target for decelerating the progression of
AD and other tauopathy-related neurodegenerative diseases.
EXPERIMENTAL SECTION
■
General Procedures for Characterization of Compounds. All
compounds were purified by normal phase flash column chromatog-
raphy on silica gel using a CombiFlash instrument and a solvent
gradient from 5% EtOAc in hexanes to 100% EtOAc and then to 20%
MeOH in EtOAc, unless otherwise indicated. The homogeneity of all
final compounds (7−12) was confirmed to ≥95% by reverse-phase
HPLC. Only phosphonate esters with homogeneity ≥95% were
processed further to the final phosphonic acid inhibitors 7−9, 11, and
12. HPLC analysis was performed using a Waters ALLIANCE
instrument (e2695 with 2489 UV detector and 3100 mass
CONCLUSIONS
■
In our search for allosteric inhibitors of hFPPS, we screened a
small library of bisphosphonates using a multistage biochemical
and structural platform. Mindful of the structural features of the
allosteric pocket, we anticipated that binding in this pocket of
large lipophilic bisphosphonates may be possible, particularly in
the absence of Mg2+ ions. These studies led to the identification
of thienopyrimidine-based monophosphonate allosteric inhib-
itors. Our screening protocol was guided by DSF, ITC, 1H line-
broadening NMR, and X-ray crystallography in the presence
and absence of the enzyme’s metal cofactor. We also
characterized the binding interactions between the hFPPS
allosteric pocket and the bisphosphonate inhibitor 6a and
compared them to those observed with the selective allosteric
inhibitor 7. The protein−ligand interactions observed in both
complexes were virtually identical, thus validating screening
hFPPS under Mg2+-free conditions. Although Mg2+-free
conditions are not biologically relevant, this approach proved
to be useful for screening hFPPS and may be applicable to
other structurally/functionally related targets. Recently, a
similar three-stage biophysical screening method was reported
as a general tool for screening in fragment-based drug
discovery.29
1
spectrometer). Each final compound was fully characterized by H,
13C, and 31P NMR and HRMS. Chemical shifts (δ) are reported in
ppm relative to the internal deuterated solvent (1H, 13C) or external
H3PO4 (δ 0.00 31P), unless indicated otherwise. The high-resolution
MS spectra of final products were recorded using electrospray
ionization (ESI ) and Fourier transform ion cyclotron resonance
mass analyzer (FTMS). Method (homogeneity analysis using a Waters
Atlantis T3 C18 5 μm column): solvent A, H2O, 0.1% formic acid;
solvent B, CH3CN, 0.1% formic acid; mobile phase, linear gradient
from 95%A and 5%B to 5%A and 95%B in 13 min, then 2 min at 100%
B; flow rate, 1 mL/min.
General Synthetic Protocols. Suzuki cross-coupling reactions
and deprotection of the diethyl phosphonates to the phophonic acids
were carried out using the general protocols previously reported.12
General Protocol for SNAr displacement of the C-4 Chloro.
For the preparation of inhibitors 7, 11, and 12, this reaction was
carried out using the 4-cholo intermediate 14, whereas for the
synthesis of inhibitors 8 and 10, this reaction was carried out using the
common building block 4-chloro-6-(p-tolyl)thieno[2,3-d]pyrimidine
(formed after Suzuki cross-coupling of the 4-tolylboronic acid with
14). In a pressure vessel, the 4-chlorothienopyrimidine was dissolved
in dioxane, the amine reagent (aminophosphonate; 1.5 equiv) and
Et3N (5 equiv) were added, and the pressure vessel was sealed. The
reaction mixture was stirred at 100 °C for 15−24 h; completion of the
reaction was monitored by HPLC. The reaction mixture was cooled to
RT and diluted with ethyl acetate (50 mL). The organic layer was
washed with an aqueous solution of saturated NaHCO3 (15 mL),
water (45 mL), and brine (15 mL) and dried over anhydrous MgSO4.
The crude was purified by column chromatography to give the desired
products. The same procedure was used for the synthesis of
compound 10, however, the solvent was changed to dioxane:H2O
(1:1) and Na2CO3 (1.5 equiv) was used as the base; the reaction was
completed in 3 h.
In addition, analysis of our collective X-ray data on
thienopyrimidine-based inhibitor bound in the allylic subpocket
and the allosteric pocket (e.g., 6a(act) vs 6a(allo); PDB codes 4JVJ
and 4LPG, respectively) revealed very similar interactions
involved in both binding sites. For example, the π-stacking
interactions between the tolyl side chain of 6a and Phe99/
Gln171 in the hFPPS/6a(act) complex are analogous to those
observed in the hFPPS/6a(allo) complex with Phe206/Asn59
(Figure 5b). This finding is intriguing and supports the
hypothesis that the allosteric pocket plays a regulatory role in
product feedback inhibition that is sensitive to the concen-
tration of isoprenoids and independent of the Mg2+ cofactor
concentration.
(((6-(p-Tolyl)thieno[2,3-d]pyrimidin-4-yl)amino)methyl)-
phosphonic Acid (7). The final compound 7 was isolated as a white
1
powder (7.4 mg, 50% isolated yield). H NMR (500 MHz, D2O, 5%
We identified thienopyrimidine-based monophosphonates
(ThP-MP) as a new chemotype of hFPPS allosteric inhibitors.
These compounds are amenable to further optimization and
may provide tools for validating hFPPS as a therapeutic target
for nonskeletal diseases such as various cancers and neuro-
degenerative diseases. A relationship between the latter and the
levels of FPP/GGPP in the human brain has been previously
reported.8 Herein we provide further evidence of a genetic link
between hFPPS and the levels of phosphorylated tau (P-Tau)
in the human AD brain. The accumulation of P-Tau in the
brain is associated with the pathological accumulation of
ND4OD) δ 7.92 (s, 1H), 7.35 (s, 1H), 7.21 (d, J = 8.0 Hz, 2H), 6.90
(d, J = 8.0 Hz, 2H), 3.29 (d, J = 13.3, 2H), 2.10 (s, 3H). 13C NMR
(126 MHz, D2O, 5% ND4OD) δ 162.5, 156.4 (d, J = 9 Hz), 152.4,
140.0, 138.8, 129.6, 129.3, 125.2, 118.3, 113.2, 40.4 (d, J = 135 Hz),
20.4. 31P NMR (202 MHz, D2O, 5% ND4OD): δ 12.96. HRMS
(ESI−) m/z 334.0415 calculated for C14H13N3O3PS; found m/z
334.0420 [M − H]−.
(2-((6-(p-Tolyl)thieno[2,3-d]pyrimidin-4-yl)amino)ethyl)-
phosphonic Acid (8). The diethyl ester of inhibitor 8 was isolated as a
1
brown solid (45.8 mg, 59% yields). H NMR (500 MHz, CD3OD) δ
8.29 (s, 1H), 7.57 (s, 1H), 7.51 (d, J = 8.0 Hz, 2H), 7.21 (d, J = 8.0
Hz, 2H), 4.13−4.09 (m, 4H), 3.83−3.77 (m, 2H), 2.34 (s, 3H), 2.31−
H
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Journal of Medicinal Chemistry
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2.24 (m, 2H), 1.31 (t, J = 7.1 Hz, 3H). 13C NMR (126 MHz, CD3OD)
δ 164.0, 156.5, 153.0, 140.4, 138.6, 130.5, 129.4, 125.5, 118.1, 112.7,
62.0 (d, J = 6.5 Hz), 34.6 (d, J = 2.1 Hz), 24.6 (d, J = 138.0 Hz), 19.8,
15.3 (d, J = 6.1 Hz). 31P NMR (81 MHz, CD3OD) δ 30.33. MS (ESI
+) m/z 405.90 [M + H]+ for C19H25N3O3PS. The final inhibitor 8 was
118.1, 114.0, 110.0, 55.5 (d, J = 134 Hz), 18.6. 31P NMR (81 MHz,
D2O, 5% ND4OD) δ 13.84. HRMS (ESI−) m/z 444.0344 calculated
for C20H16ClN3O3PS; found m/z 444.0352 [M − H]−.
6-Bromo-4-chlorothieno[2,3-d]pyrimidine (14). Thieno[2,3-d]-
pyrimidin-4(3H)-one (13) was prepared from 2,5-dihydroxy-1,4-
dithiane as previously reported,16 with the exception that all reactions
were carried out under thermal conditions. Conversion of intermediate
13 to 14 was achieved by successive reaction of 13 with Br2 and POCl3
as previously reported.30 The 6-bromothieno[2,3-d]pyrimidin-4(3H)-
one intermediate was obtained as a light-brown solid in 63% yield. The
1H NMR data was consistent with the literature: 1H NMR (300 MHz,
DMSO-d6) δ 12.64 (bs, 1H), 8.15 (s, 1H), 7.56 (s, 1H)]. The 6-
bromo-4-chlorothieno[2,3-d]pyrimidine (14) was obtained as a yellow
1
obtained as a light-brown solid (26.2 mg, near quantitative yield). H
NMR (500 MHz, D2O, 5% ND4OD) δ 7.92 (s, 1H), 7.14 (d, J = 8.0
Hz, 2H), 7.09 (s, 1H), 6.88 (d, J = 8.0 Hz, 2H), 3.50−3.45 (m, 2H),
2.13 (s, 3H), 1.76−1.73 (m, 2H). 13C NMR (126 MHz, D2O, 5%
ND4OD) δ 162.4, 155.6, 152.2, 139.9, 138.6, 129.4, 129.2, 124.9,
118.0, 112.9, 37.7, 29.2 (d, J = 126 Hz), 20.4. 31P NMR (202 MHz,
D2O, 5% ND4OD): δ 18.5. HRMS (ESI−) m/z 348.0577 calculated
for C15H15N3O3PS; found m/z 348.0569 [M − H]−.
1
(2-(6-(p-Tolyl)thieno[2,3-d]pyrimidin-4-yl)ethyl)phosphonic Acid
(9). A solution of tetraethyl methylenediphosphonate (26 μL, 0.104
mmol) in THF (3 mL) was cooled to 0 °C, and NaH (60%, 5 mg,
0.125 mmol) was added in a portion and then stirred at 0 °C for 15
min. A solution of aldehyde 15 (29 mg, 0.114 mmol) in THF (1 mL)
was added, and the reaction mixture was stirred at RT for 1 h. The
reaction was quenched with H2O, diluted with EtOAc (100 mL), and
extracted with 1 N HCl solution (50 mL) and brine (100 mL). The
organic layer was collected, dried over MgSO4, concentrated, and
purified by chromatography on silica gel (solvent gradient from 0% to
100% EtOAc in Hex). The isolated product was dissolved in EtOH (3
mL), 10% Pd/C (5 mg) was added, and the reaction mixture was
stirred under an atmosphere of H2 for 1 h. The reaction mixture was
filtered through Celite and washed with MeOH. The filtrate was
concentrated to isolate the product. The isolated product was
redissolved in DCM (3 mL) and TMSBr (150 μL, 0.127 mmol)
was added dropwise. The reaction mixture was stirred at RT overnight.
Subsequently, MeOH (2 mL) was added to the reaction mixture, and
stirring was continued for an additional 1 h. The solvent was
evaporated under vacuum, and the residue was redissolved in MeOH
and triturated with DCM to precipitate the final product. After
filtration, compound 9 was isolated as white solid (45% overall yield
solid in 58% yield. The H NMR data was also consistent with the
literature: 1H NMR (500 MHz, CDCl3) δ 8.81 (s, 1H), 7.48 (s, 1H)].
6-(p-Tolyl)thieno[2,3-d]pyrimidine-4-carbaldehyde (15). Step f
(Scheme 1). The common building block 4-chloro-6-(p-tolyl)thieno-
[2,3-d]pyrimidine (50 mg, 0.19 mmol, 1 equiv) was mixed with
potassium vinyltrifluoroborate (28 mg, 0.21 mmol, 1.1 equiv) and
PdCl2(dppf)CH2Cl2 (8 mg, 0.01 mmol, 0.05 equiv) and purged with
argon. i-PrOH/H2O (2 mL, 2/1 ratio) and Et3N (58 mg, 0.58 mmol, 3
equiv) were added, and the mixture was purged again with argon. The
mixture was sealed in pressure vessel and was heated at 100 °C for 1 h.
The reaction mixture was cooled to RT, then filtered through Celite,
washed with EtOAc, concentrated, and purified by chromatography
(100% Hex to 20% EtOAc in Hex) on silica gel to give the C-4 vinyl
intermediate 6-(p-tolyl)-4-vinylthieno[2,3-d]pyrimidine as yellow solid
1
(35 mg; 72% yield). The H NMR and MS were consistent with the
1
desired product. H NMR (CDCl3): δ 2.40 (s, 3H, −CH3), 5.85 (d, J
= 10.70 Hz, 1 H), 6.77 (d, J = 10.70 Hz), 7.16−7.23 (m, 1H), 7.26 (d,
J = 8.10 Hz, 2H), 7.57 (s, 1H), 7.60 (d, J = 8.10 Hz, 2H), 8.97 (s, 1H).
MS (ESI+) m/z 255.1 [M + H]+ for C15H14N2S.
Step g (Scheme 1). To a solution of the above 4-vinyl-
thienopyrimidine (50 mg, 0.2 mmol) in acetone:H2O (4 mL; 10:1
ratio), 2,6-lutidine (42 mg, 0.4 mmol, 2 equiv), 4-methylmorpholine-
N-oxide (35 mg, 0.3 mmol, 1.5 equiv), and osmium tetroxide (0.1 mL
of a 40.4 mM solution in toluene) were added. The mixture was stirred
for 2 h at RT (at which point LCMS indicated complete conversion to
the desirable diol). Then, 1 mL of H2O was added followed by NaIO4
in small portions, and the mixture was stirred at RT for 1 h. The
reaction was quenched with saturated aqueous solution of sodium
thiosulfate (10 mL), and the mixture was extracted with ethyl acetate
(3 × 15 mL), washed with saturated aqueous solution of ammonium
chloride, dried over anhydrous MgSO4, and concentrated under
vacuum. The crude residue was purified by flash column
chromatography on silica gel eluted with hexane−ethyl acetate (7:1)
to give the aldehyde 15 as a yellow solid (32 mg, 64% yield). 1H NMR
(CDCl3) δ: 2.43 (s, 3H), 7.30 (d, J = 8.00 Hz, 2H), 7.70 (d, J = 8.10
Hz, 2H), 8.31 (s, 1H), 9.24 (s, 1H), 10.25 (s, 1H). 13C NMR (125
MHz, CDCl3) δ 194.20, 171.65, 152.77, 150.54, 149.08, 140.65,
129.98, 129.81, 128.84, 127.01, 114.19, 21.40. MS (ESI+) m/z 255.12
[M + H]+ for C14H11N2OS.
1
for the three steps). H NMR (500 MHz, D2O, 5% ND4OD) δ 8.50
(s, 1H), 7.63 (s, 1H), 7.30 (d, J = 8.6 Hz, 2H), 6.89 (d, J = 8.6 Hz,
2H), 3.07−3.12 (m, 1H), 2.15 (s, 3H), 1.60−1.80 (m, 2H). 13C NMR
(125 MHz, D2O, 5% ND4OD) δ 166.46, 165.63, 150.84, 144.05,
139.43, 130.99, 129.17, 128.89, 125.61, 114.32, 30.81, 28.93 (d, J =
129.6 Hz), 20.36. 31P NMR (162 MHz, D2O, 5% ND4OD) δ 19.53.
HRMS (ESI−) m/z 333.0541 calculated for C15H14N2O3PS; found m/
z 333.0473 [M − H]−.
2-((6-(p-Tolyl)thieno[2,3-d]pyrimidin-4-yl)amino)acetic Acid (10).
The compound was isolated as a white solid (13.8 mg, 60% yield). 1H
NMR (500 MHz, DMSO-d6) δ 8.34 (s, 1H), 7.99 (s, 1H), 7.58 (d, J =
8.1 Hz, 2H), 7.32 (d, J = 8.1 Hz, 2H), 5.76 (s, 1H), 4.18 (d, J = 4.7 Hz,
2H), 2.35 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ 171.5, 164.8,
156.5, 153.6, 138.8, 138.3, 130.4, 130.0, 125.6, 117.7, 114.4, 55.0, 20.8.
HRMS (ESI−) m/z 298.0656 calculated for C15H12N3O2S; found m/z
298.0654 [M − H]−.
(((6-(3-Chloro-4-methylphenyl)thieno[2,3-d]pyrimidin-4-yl)-
amino)methyl)phosphonic Acid (11). Compound 11 was isolated as
a pale-yellow powder (8.5 mg, 25% overall isolated yield for the last
Diethyl (Amino(phenyl)methyl)phosphonate (16b). In a round-
bottom flask, benzaldehyde (742.79 mg, 7.00 mmol, 1 equiv) was
mixed with magnesium perchlorate (156.23 mg, 0.7 mmol, 0.1 equiv)
for 15 min. Benzylamine (750 mg, 7.00 mmol, 1 equiv) and
diethylphosphite (0.939 mL, 7.28 mmol, 1.04 equiv) were added,
and the reaction mixture was heated at 85 °C for 24 h. The crude
product was dried under vacuum and purified by column
chromatography on silica gel to give the diethyl ((benzylamino)-
(phenyl)methyl) phosphonate product as a slightly yellow oil in 81%
1
two steps). H NMR (400 MHz, D2O, 5% ND4OD) δ 8.05 (s, 1H),
7.42 (s, 1H), 7.28 (s, 1H), 7.23 (d, J = 8.0 Hz, 1H), 7.05 (d, J = 8.0
Hz, 1H), 3.44 (d, J = 13.2 Hz, 2H), 2.19 (s, 3H). 13C NMR (126
MHz, D2O, 5% ND4OD) δ 162.6, 156.4, 152.5, 138.2, 136.0, 134.0,
131.5, 131.0, 125.0, 123.5, 118.0, 114.2, 40.3 (d, J = 134 Hz), 19.0. 31P
NMR (202 MHz, D2O, 5% ND4OD): δ 13.67. HRMS (ESI−) m/z
368.0031 calculated for C14H12ClN3O3PS; found m/z 368.0030 [M −
H]−.
1
yield (1.8 g). H NMR (300 MHz, CDCl3) 7.43−7.28 (m, 10H),
4.15−3.80 (m, 7H), δ 3.56 (d, J = 13.3 Hz, 1H), 2.12 (s, 1H), 1.29 (t, J
= 7.0 Hz, 3H), 1.14 (t, J = 7.0 Hz, 3H).31 Hydrogenation of the diethyl
((benzylamino)(phenyl)methyl)phosphonate intermediate (200 mg,
0.6 mmol) in 4.4% formic acid in methanol (6 mL) using Pearlman’s
catalyst (84.25 mg, 0.6 mmol, 0.2 equiv) under argon for 2 h gave the
desired crude product,32 which was purified by column chromatog-
raphy on silica gel to give the free amine as a transparent oil (145.9
mg, quantitative yield). 1H NMR (500 MHz, CDCl3) δ 7.45 (d, J = 7.7
(((6-(3-Chloro-4-methylphenyl)thieno[2,3-d]pyrimidin-4-yl)-
amino)(phenyl)methyl)phosphonic Acid (12). Compound 12 was
isolated as a white solid (20.8 mg, 48% yield for the last two steps). 1H
NMR (500 MHz, D2O, 5% ND4OD) δ 7.77 (s, 1H), 7.49 (d, J = 7.7
Hz, 3H), 7.35 (t, J = 7.2 Hz, 2H), 7.25, −7.22 (m, 1H), 7.05 (s, 1H),
6.79 (s, 1H), 6.27 (d, J = 7.2 Hz, 1H), 5.00 (d, J = 19.7 Hz, 1H), 1.75
(s, 3H). 13C NMR (126 MHz, D2O, 5% ND4OD) δ 162.8, 155.8,
152.2, 140.9, 138.4, 135.6, 131.2, 128.1, 127.8, 126.6, 124.8, 123.2,
I
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Journal of Medicinal Chemistry
Article
Hz, 2H), 7.35 (t, J = 7.7 Hz, 2H), 7.30 (dd, J = 7.3, 2.0 Hz, 1H), 4.26
(d, J = 17.1 Hz, 1H),4.08−402 (m, 2H), 4.02−3.94 (m, 1H), 3.90−
3.84 (m, 1H), 1.92 (br s, 2H), 1.27 (t, J = 7.0 Hz, 3H), 1.18 (t, J = 7.0
Hz, 3H).
mercaptoethanol, and 5% glycerol) at 50 μM and 1 mM
concentrations, respectively. Each experiment consisted of a first 1
μL injection of the ligand followed by 18 2 μL injections into the cell
containing 200 μL of the protein sample. Heats of dilution were
measured in control titrations and subtracted from the actual data. The
data were analyzed with the Origin 7 software provided with the
instrument.
Diethyl (((6-Bromothieno[2,3-d]pyrimidin-4-yl)amino)methyl)-
phosphonate (17a; R2 = H). The product was isolated as yellow
1
solid (884 mg, 50% yield). H NMR (400 MHz, CD3OD): δ 8.36 (s,
1H), 7.56 (s, 1H), 4.20−4.12 (m, 6H), 1.28 (t, J = 7.1 Hz, 6H). 13C
NMR (75 MHz, CD3OD): δ 167.7, 157.0 (d, J = 1.7 Hz), 154.8, 122.8,
118.8, 112.7, 64.1 (d, J = 6.7 Hz), 36.7 (d, J = 158 Hz), 16.7 (d, J = 5.9
Hz). 31P NMR (81 MHz, CD3OD): δ 23.91. MS (ESI+) m/z 380.10
[M + H]+ for C11H15BrN3O3PS.
1
Evaluation of Competitive Binding by H Line Broadening
NMR. Spectra were acquired on a 500 MHz Varian INOVA
instrument, with a HCN cold probe and z-axis pulsed-field gradients,
1
at the Quebec/Eastern Canada High Field NMR Facility. H NMR
spectra were acquired with a 1D presaturation sequence, with a sweep
width of 8000 Hz and an acquisition time of 1 s. A low power
saturation pulse was applied at the HOD frequency during the 3 s
relaxation delay. A 5 mm NMR tube was charged with 540 μL of D2O
and 6 μL of an inhibitor (10 mM stock solution in H2O), and the tube
was briefly mixed using a vortex. The 1H NMR spectrum was acquired
and the water-suppression parameters were determined (presatura-
tion). Buffer solution (60 μL) was added to the NMR tube and briefly
mixed for a final inhibitor concentration of 100 μM in buffer
containing 50 mM Tris buffer at pH 7.7, 2 mM MgCl2, 0.5 mM TCEP,
Diethyl (((6-Bromothieno[2,3-d]pyrimidin-4-yl)amino)(phenyl)-
methyl)phosphonate (17b; R2 = Ph). The product was isolated as a
1
white solid (51 mg, 36% yield). H NMR (300 MHz, CDCl3) δ 8.42
(s, 1H), 8.11−8.07 (m, 1H), 7.70−7.67 (m, 3H), 7.27−7.22 (m, 2H),
6.32 (dd, J = 22.4, 9.6 Hz, 1H), 4.31−4.07 (m, 3H), 3.93−3.84 (m,
1H), 1.28−1.19 (s, 6H). 13C NMR (75 MHz, CDCl3) δ 167.7, 155.0
(d, J = 9 Hz), 153.7, 128.49 (d, J = 2 Hz), 128.44 (d, J = 2 Hz), 127.99
(d, J = 3 Hz), 121.7, 117.8, 111.3, 63.5 (d, J = 7 Hz), 50.79 (d, J = 156
Hz), 16.3 (dd, J = 19, 6 Hz). MS (ESI+) m/z 456.14 [M + H]+ for
C17H21BrN3O3PS
1
and 20 μg/mL BSA. The H NMR spectrum was acquired of the free
Diethyl (((6-(p-Tolyl)thieno[2,3-d]pyrimidin-4-yl)amino)methyl)-
inhibitor. Aliquots of the hFPPS solution in buffer (containing 22.63
μg/μL protein, 50 mM Tris at pH 7.7, 500 mM NaCl, 5% glycerol,
and 0.5 mM TCEP) were added, followed by gentle mixing, to bring
the inhibitor to hFPPS ratio as indicated on each Figure. Finally, a
second inhibitor or IPP (purchased from Isoprenoids, LC, as its
ammonium salt; 40 mM solution was prepared in 0.5 mM Tris buffer
at pH 7.7) was added in incremental amounts to obtain the indicated
ratios and confirm whether binding of the two inhibitors was
competitive or not (usually titration with the second inhibitor was
continued until the resonances of the first inhibitor were restored, or
up to the molar ratio of hFPPS: inhibitor A; inhibitor B indicated on
the NMR spectra).
Co-crystallization of hFPPS with Inhibitors 6a and 7 in the
Absence of Magnesium Ions. Compounds 6a and 7 were prepared
as 100 and 25 mM stock solutions in 100 mM TrisHCl (pH 7.5)
buffer, respectively. Each stock solution was added to the purified
hFPPS sample to give the final concentrations of 5 mM inhibitor and
0.25 mM protein. A microseeding technique was employed to obtain
crystals suitable for X-ray diffraction analysis. Crystals were grown at
22 °C by vapor diffusion in sitting drops composed of 1 μL of protein/
inhibitor mixture and 1 μL of crystallization buffer, and an additional
0.5 μL of seed solution when added. Seed solutions were prepared
with Seed Bead kits (Hampton Research). For the hFPPS/6a(allo)
complex, the initial crystals formed in a crystallization solution
composed of 0.1 M TrisHCl (pH 8.5) and 2 M ammonium
dihydrogen phosphate. Diffraction quality crystals were obtained in a
seeding optimization trial with a new crystallization buffer composed
of 0.085 M HEPES (pH 7.5), 17% (w/v) PEG 10K, 6.8% (v/v)
ethylene glycol, and 15% (v/v) glycerol. For the hFPPS/7 complex, a
single crystal was obtained under the same condition, but with
microseeds prepared from the hFPPS/6a crystals.
phosphonate (18a; R2 = R3 = H). The product was isolated as a
1
white solid (25.5 mg, 65% yields). H NMR (500 MHz, CD3OD): δ
8.35 (s, 1H), 7.68 (s, 1H), 7.56 (d, J = 8.1 Hz, 2H), 7.25 (d, J = 8.1
Hz, 2H), 4.20−4.14 (m, 6H), 2.36 (s, 3H), 1.29 (t, J = 7.1 Hz, 6H).
13C NMR (126 MHz, CD3OD): δ 164.4, 156.4, 152.8, 140.8, 138.8,
130.5, 129.4, 125.6, 118.2, 112.7, 62.7 (d, J = 6.7 Hz), 35.3 (d, J =
158.1 Hz), 19.8, 15.3 (d, J = 5.9 Hz). 31P NMR (81 MHz, CD3OD):

δ 25.16. MS (ESI+) m/z 391.9 [M + H]+ for C18H23N3O3PS.
Diethyl (((6-(3-Chloro-4-methylphenyl)thieno[2,3-d]pyrimidin-4-
yl)amino)methyl)phosphonate (18b; R2 = H, R3 = Cl). The product
was isolated as a pale-orange solid (9.6 mg, 29% yield). 1H NMR (500
MHz, CD3OD) δ 8.38 (s, 1H), 7.76 (s, 1H), 7.39 (t, J = 9.4 Hz, 2H),
7.32 (t, J = 7.9 Hz, 1H), 4.21−4.14 (m, 6H), 2.30 (s, 3H), 1.29 (t, J =
7.1 Hz, 6H). 31P NMR (202 MHz, CD3OD) δ 23.67. MS (ESI+) m/z
426.3 [M + H]+ for C18H22ClN3O3PS.
Diethyl (((6-(3-Chloro-4-methylphenyl)thieno[2,3-d]pyrimidin-4-
yl)amino)(phenyl)methyl)phosphonate (18c; R2 = Ph, R3 = Cl).
The product was isolated as a pale-yellow solid (27.2 mg, 60% yield).
1H NMR (400 MHz, CDCl3) δ 8.46 (s, 1H), 7.74 (s, 1H), 7.72 (s,
2H), 7.53 (d, J = 1.6 Hz, 1H), 7.31−7.20 (m, 5H), 6.32 (d, J = 22.0
Hz, 1H), 4.31−4.14 (m, 2H), 4.02−3.93 (m, 1H), 3.78−3.68 (m, 1H),
2.39 (s, 3H), 1.23 (t, J = 7.0 Hz, 3H), 1.06 (t, J = 7.0 Hz, 3H). 13C
NMR (75 MHz, CDCl3) δ 166.4, 155.9 (d, J = 9 Hz), 153.6, 138.9,
136.3, 135.5, 134.9, 133.0, 131.3, 128.6, 128.6, 128.1 (d, J = 3 Hz),
126.6, 124.6, 118.3, 114.3, 63.5 (d, J = 7 Hz), 51.8 (d, J = 155 Hz),
49.8, 19.9, 16.4 (d, J = 6 Hz), 16.0 (d, J = 6 Hz). 31P NMR (81 MHz,
CDCl3) δ 21.64. MS (ESI+) m/z 502.25 [M + H]+ for
C24H26ClN3O3PS.
In Vitro Inhibition Assays. In vitro enzymatic assays were carried
out using method 2 (M2) as previously described.12d
Differential Scanning Fluorimetry Studies. DSF studies were
carried out using the general conditions previously described.1c
Samples were prepared to a final volume of 25 μL containing 4 μM
hFPPS protein, 10 mM HEPES (pH 7.5), 10 mM NaCl, with or
without 5 mM MgCl2, and 5× SYPRO Orange dye (diluted from
commercial stock solution of 5000×; Invitrogen). All samples were
prepared in triplicate. Fluorescence was measured using an iCycler
RT-PCR instrument with an iQ5 detector (Bio-Rad) while increasing
the temperature gradient from 30 to 90 °C in increments of 0.5 °C/10
s. The midpoint temperature of the unfolding protein transition (Tm)
was calculated using the software package from Bio-Rad iQ5. The
inhibitor and substrate concentrations for each experiment are
indicated in the figures.
X-ray Data Collection, Processing, and Structure Refine-
ment. For structure determination, diffraction data were collected
from single crystals at 100 K with synchrotron radiation (Canadian
Light Source, Saskatoon, SK) and a Rayonix MX300 CCD detector.
The data were processed with the xia2 package.33 The initial structure
models were built by difference Fourier methods with a ligand/
solvent-omitted input model generated from the PDB entry 2F7M.
The models were improved through iterative rounds of manual and
automated refinement with COOT34 and REFMAC5.35 The final
models were deposited into the Protein Data Bank. To measure the
anomalous signal from the phosphorus and sulfur atoms in the
hFPPS/6a(allo) complex, additional data were collected from a single
crystal at 100 K with a MicroMax-007 HF generator (Rigaku) and a
Saturn 944+ CCD detector (Rigaku). This data set was also processed
with the xia2 package but without merging the Friedel mates. An
anomalous density map was calculated from the processed data with
the programs SHELXC36 and ANODE.37 Data collection and
Isothermal Titration Calorimetry (ITC) Studies. ITC experi-
ments were carried out at 30 °C with a MicroCal iTC200 system from
GE Healthcare. The protein and ligand solutions were prepared in the
same buffer (10 mM HEPES (pH 7.5), 500 mM NaCl2, 2 mM β-
J
dx.doi.org/10.1021/jm500629e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 3. Data Collection and Refinement Statistics
data set 1 (synchrotron) inhibitor 6a, Pi
data set 2 (home source) inhibitor 6a, Pi
data set 3 (synchrotron) inhibitor 7, Pi
PDB ID
4LPG
4LPH
Data Collection
1.5418
wavelength (Å)
space group
0.979 49
0.979 49
P41212
P41212
P41212
unit cell (Å)
a = b = 110.87, c = 77.03
49.58−2.35 (2.41−2.35)
14.4 (14.4)
a =b = 110.96, c = 76.97
39.23−2.61 (2.67−2.61)
7.2 (4.7)
a = b = 111.12, c = 77.19
55.06−2.30 (2.36−2.30)
9.4 (9.5)
a
resolution (Å)
redundancy
completeness (%)
I/σ(I)
99.7 (98.0)
98.7 (99.0)
98.4 (99.1)
36.7 (5.4)
39.3 (5.5)
22.3 (4.8)
Rmerge
0.044 (0.527)
0.055 (0.411)
Refinement
no. reflections
19406
2666
20290
2700
no. protein atoms
no. ligand atoms
no. solvent atoms
31
27
40
50
R
work/Rfree
0.182/0.230
0.183/0.226
average B factor (Å2)
protein atoms
73.9
80.4
78.9
93.4
ligand atoms
rms deviations
bond length (Å)
bond angle (deg)
0.016
1.8
0.017
1.8
a
Values in parentheses are for the highest resolution shell.
refinement statistics, as well as the PDB IDs for the structure models,
are presented in Table 3.
streptavidin. Samples were subsequently incubated with tetramethyl-
benzidine and 0.006% hydrogen peroxide per manufacturer’s
instructions. The reaction was stopped with diluted sulfuric acid and
optical density measurements read using a Molecular Devices
Spectramax Plus plate reader. Intra-assay and interassay variability
measures were 5.1% and 9.1%, consistent with manufacturer
recommendation.
Lactate Dehydrogenase Assay (LDH). Lactate dehydrogenase
activity was measured using a commercial kit. Maximum toxicity was
based on % lactic acid dehydrogenase activity observed in the medium
of cells treated with an hFPPS inhibitor as compared to the control
(untreated cells); activity observed in the untreated control cells was
set to 0%.
Studies on Genetic Associations. Using a cohort of 751 pairs of
Alzheimer and age-matched control French Canadian subjects from a
population isolate from eastern Canada, we performed the mapping of
some 535000 polymorphisms in each of our case and control subjects
using the Illumina 550k Human Quad genomic beadchip according to
methodology reported previously.38 Five single nucleotide poly-
morphisms (SNPs) were examined in the human FPPS gene loci. A
specific genetic associations between variant rs4971072 (A/G) in the
promoter area of hFPPS and Tau protein levels (p < 0.02) in the
cortical area was found to be significantly associated with phospho-tau
concentrations (p < 0.02) in the same brain region in the AD cohort.
Analysis of mRNA Prevalence. A subset of AD (n = 34) and age-
matched control brains (n = 24) were used to quantify the mRNA
prevalence of hFPPS using real time quantitative polymerase chain
reaction (QrtPCR) as adapted from our previous studies.28
Total Tau Protein and Phospho-Tau Levels in the
Alzheimer’s Brain and in Response to hFPPS Inhibitors. Frontal
cortex brain samples from autopsy-confirmed cases of Alzheimer’s
disease and age-matched control subjects were obtained from the
Douglas Institute Brain Bank, Montreal, Canada. Frozen brain samples
(n = 116) were homogenized and prepared according to a previously
established protocol27 prior to ELISA quantification for total cortical
tau protein and phospho-tau levels. Human neuroblastoma cell
cultures (SH-Sy5y from the ATCC collection) were used to assay
the effects of our newly developed hFPPS inhibitors on tau
metabolism and general toxicity. Typically, cells were grown in
serum condition for 5 days and then exposed to different
concentrations (1−500 nM) of our inhibitors for a period of 24 h.
Cell were washed and lysed prior to tau and phospho-tau ELISA
quantification. Total tau (T-Tau) and phospho-tau (P-Tau)
concentrations were measured using a commercial enzyme immuno-
assay (Innotest Inc., Ghent, Belgium). In this assay, the wells of
polystyrene microtiter plates were coated with the solid phase anti-
human tau monoclonal antibody (AT120 for tau and AT270 for
phospho-tau). The test samples were incubated in these wells along
with two separate biotinylated tau monoclonal antibodies (H57 and
BT2) that recognize different tau and P-Tau epitopes. Samples were
rinsed with an assay buffer and then incubated with peroxidase-labeled
ASSOCIATED CONTENT
■
S
* Supporting Information
1
NMR spectra and homogeneity data for inhibitors 7−12. H
NMR competition studies and X-ray data. Genetic analysis on
hFPPS polymorphisms. This material is available free of charge
via the Internet at http://pubs.acs.org.
Accession Codes
The following codes have been deposited in the Protein Data
Bank: 4LPG (hFPPS/6a/Pi complex) and 4LPH (hFPPS/7/Pi
complex).
AUTHOR INFORMATION
■
Corresponding Author
*Phone 514-398-3638. Fax 514-398-3797. E-mail: Youla.
tsantrizos@mcgill.ca.
Author Contributions
#J. W. De Schutter and J. Park contributed equally to the work
described in this manuscript.
Notes
The authors declare no competing financial interest.
∇Zheping Hu: Undergraduate Research Participant.
K
dx.doi.org/10.1021/jm500629e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
the Medical Research Council Myeloma IX Trial. Blood 2012, 119,
5374−5383. (b) Gnant, M.; Mlineritsch, B.; Schippinger, W.; Luschin-
ACKNOWLEDGMENTS
■
We thank Dr. Alexios Matralis for contributing to the synthesis
Ebengreuth, G.; Postlberger, S.; Menzel, C.; Jakesz, R.; Seifert, M.;
̈
of some key building blocks. 1H NMR experiments were
Hubalek, M.; Bjelic-Radisic, V.; Samonigg, H.; Tausch, C.; Eidtmann,
H.; Steger, G.; Kwasny, W.; Dubsky, P.; Fridrik, M.; Fitzal, F.; Stierer,
́
acquired on a 500 MHz NMR instrument at the Quebec/
Eastern Canada High Field NMR Facility, supported by the
Natural Sciences and Engineering Research Council of Canada
(NSERC), the Canada Foundation for Innovation (CFI), the
M.; Rucklinger, E.; Greil, R. Endocrine therapy plus zoledronic acid in
̈
premenopaisal breast cancer. New Engl. J. Med. 2009, 360, 679−691.
(c) Coleman, R. E.; Marshall, H.; Cameron, D.; Dodwell, D.;
Burkinshaw, R.; Keane, M.; Gil, M.; Houston, S. J.; Grieve, R. J.;
Barrett-Lee, P. J.; Ritchie, D.; Pugh, J.; Gaunt, C.; Rea, U.; Peterson, J.;
Davies, C.; Hiley, V.; Gregory, W.; Bell, R. Breast-cancer adjuvant
therapy with zoledronic Acid. New Engl. J. Med. 2011, 365, 1396−
1405.
́ ̀
Quebec Ministere de la Recherche en Science et Technologie
(FQRNT), and McGill University. Financial support for this
work was provided by NSERC and FQRNT (research grants to
Y. S. Tsantrizos) and the Canadian Institute of Health Research
(CIHR; research grants to A. M. Berghuis, J. Poirier, and Y. S.
Tsantrizos).
(7) Morita, C. T.; Jin, C.; Sarikonda, G.; Wang, H. Nonpeptide
antigens, presentation mechanisms, and immunological memory of
human Vγ2Vδ2 T cells: discriminating friends from foe through the
recognition of prenyl pyrophosphate antigens. Immunol. Rev. 2007,
215, 59−76.
ABBREVIATIONS USED
■
hFPPS, human farnesyl pyrophosphate synthase; hGGPPS,
human geranylgeranyl pyrophosphate synthase; hSQS, human
squalene synthase; DMAPP, dimethylallyl pyrophosphate; IPP,
isopentenyl pyrophosphate; GPP, geranyl pyrophosphate; N-
BPs, nitrogen-containing bisphosphonates; GTPases, small
guanine triphosphate binding proteins; IFNγ, interferon-
gamma; SAR, structure−activity relationship; DSF, differential
scanning fluorimetry; AD, Alzheimer’s disease; P-Tau,
phosphorylated tau; phospho-tau, phosphorylated tau; T-Tau,
total tau protein; LDH, lactate dehydrogenase
(8) (a) Eckert, G. P.; Hooff, G. P.; Strandjord, D. M.; Igbavboa, U.;
Volmer, D. A.; Muller, W. E.; Wood, W. G. Regulation of the brain
̈
isoprenoids farnesyl- and geranylgeranylpyrophosphate is altered in
male Alzheimer patients. Neurobiol. Dis. 2009, 35, 251−257.
(b) Hooff, G. P.; Wood, W. G.; Muller, W. E.; Eckert, G. P.
̈
Isoprenoids, small GTPases and Alzheimer’s diseases. Biochim. Biophys.
Acta 2010, 1801, 896−905.
(9) Jahnke, W.; Rondeau, J.-M.; Cotesta, S.; Marzinzik, A.; Pelle,
́
X.;
Geiser, M.; Strauss, A.; Gotte, M.; Bitsch, F.; Hemmig, R.; Henry, C.;
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Lehmann, S.; Glickman, J. F.; Roddy, T. P.; Stout, S. J.; Green, J. R.
Allosteric non-bisphosphonate FPPS inhibitors identified by fragment-
based discovery. Nature Chem. Biol. 2010, 6, 660−666.
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