Fluorescence probe for lysophospholipase C/NPP6 activity and a potent NPP6 inhibitor

Article abstract of DOI:10.1021/ja201028t

Nucleotide pyrophosphatases/phosphodiesterases (NPPs) are ubiquitous membrane-associated or secreted ectoenzymes that have a role in regulating extracellular nucleotide and phospholipid metabolism. Among the members of the NPP family, NPP1 and -3 act on nucleotides such as ATP, while NPP2, -6, and -7 act on phospholipids such as lysophosphatidylcholine and sphingomyelin. NPP6, a recently characterized NPP family member, is a choline-specific glycerophosphodiester phosphodiesterase, but its functions remain to be analyzed, partly due to the lack of highly sensitive activity assay systems and practical inhibitors. Here we report synthesis of novel NPP6 fluorescence probes, TG-mPC and its analogues TG-mPC₃C, TG-mPC₅C, TG-mPENE, TG-mPEA, TG-mPhos, TG-mPA, TG-mPMe, and TG-mPPr. Among the seven NPPs, only NPP6 hydrolyzed TG-mPC, TG-mPC₃C, and TG-mPENE. TG-mPC was hydrolyzed in the cell lysate from NPP6-transfected cells, but not control cells, showing that it is suitable for use in cell-based NPP6 assays. We also examined the usefulness of TG-mPC as a fluorescence imaging probe. We further applied TG-mPC to carry out high-throughput NPP6 inhibitor screening and found several NPP6-selective inhibitors in a library of about 80 000 compounds. Through structure-activity relationship (SAR) analysis, we identified a potent and selective NPP6 inhibitor with an IC₅₀ value of 0.21 μM. Our NPP6-selective fluorescence probe, TG-mPC, and the inhibitor are expected to be useful to elucidate the biological function of NPP6.

Full text of DOI:10.1021/ja201028t

ARTICLE
pubs.acs.org/JACS
Fluorescence Probe for Lysophospholipase C/NPP6 Activity
and a Potent NPP6 Inhibitor†
Mitsuyasu Kawaguchi,‡,§ Takayoshi Okabe,^ Shinichi Okudaira,# Kenjiro Hanaoka,‡,§ Yuuta Fujikawa,‡,§
Takuya Terai,‡,§ Toru Komatsu,‡,§ Hirotatsu Kojima,§,^ Junken Aoki,# and Tetsuo Nagano*,‡,§,^
‡Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
§CREST, JST, Sanbancho-blg, 5 Sanbancho, Chiyoda-ku, Tokyo, 102-0075, Japan
^Chemical Biology Research Initiative, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
#Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aoba Aramaki, Aoba-ku, Sendai, Miyagi 980-8578, Japan
S Supporting Information
b
ABSTRACT: Nucleotide pyrophosphatases/phosphodies-
terases (NPPs) are ubiquitous membrane-associated or se-
creted ectoenzymes that have a role in regulating extra-
cellular nucleotide and phospholipid metabolism. Among the
members of the NPP family, NPP1 and -3 act on nucleotides
such as ATP, while NPP2, -6, and -7 act on phospholipids such
as lysophosphatidylcholine and sphingomyelin. NPP6, a re-
cently characterized NPP family member, is a choline-specific
glycerophosphodiester phosphodiesterase, but its functions
remain to be analyzed, partly due to the lack of highly sensitive
activity assay systems and practical inhibitors. Here we report
synthesis of novel NPP6 fluorescence probes, TG-mPC and its
analogues TG-mPC3C, TG-mPC5C, TG-mPENE, TG-mPEA,
TG-mPhos, TG-mPA, TG-mPMe, and TG-mPPr. Among the
seven NPPs, only NPP6 hydrolyzed TG-mPC, TG-mPC3C, and TG-mPENE. TG-mPC was hydrolyzed in the cell lysate from
NPP6-transfected cells, but not control cells, showing that it is suitable for use in cell-based NPP6 assays. We also examined
the usefulness of TG-mPC as a fluorescence imaging probe. We further applied TG-mPC to carry out high-throughput
NPP6 inhibitor screening and found several NPP6-selective inhibitors in a library of about 80 000 compounds. Through
structureꢀactivity relationship (SAR) analysis, we identified a potent and selective NPP6 inhibitor with an IC50 value of
0.21 μM. Our NPP6-selective fluorescence probe, TG-mPC, and the inhibitor are expected to be useful to elucidate the
biological function of NPP6.
’ INTRODUCTION
as CPF4 and FS-3 were developed recently, and a number of
NPP2 inhibitors were identified using these NPP2-specific
probes.20ꢀ30 By contrast, the roles of NPP4ꢀ7 have not been
determined in detail.3 Isoform-specific fluorescence probes and
inhibitors would be useful for this purpose.
NPP6/lysoPLC is a choline-specific glycerophosphodiester
phosphodiesterase and is dominantly expressed in brain and
kidney, especially in proximal tubules. Its natural substrates
are choline-containing glycerophosphodiesters, such as lyso-
phosphatidylcholine (LPC) and glycerophosphorylcholine
(GPC).3 It has been suggested that NPP6 is important for
the reuptake of physiologically essential choline in kidney and
also in brain development. Furthermore, Cravatt et al. re-
ported that NPP6, in addition to fatty acid amide hydrolase
(FAAH), metabolizes phosphorylcholine N-acylethanolamines
Nucleotide pyrophosphatases/phosphodiesterases (NPPs)
are ubiquitous membrane-associated or secreted ectoenzymes
that have roles in regulating extracellular levels of nucleotides,
such as ATP and ADP, and phospholipids, such as lysopho-
sphatidylcholine (LPC) and sphingomyelin (SM).1ꢀ4 The mam-
malian NPP family consists of seven members (NPP1ꢀ7), which
are divided into two subgroups, NPP1ꢀ3 (type II membrane
proteins) and NPP4ꢀ7 (type I membrane proteins), based on
their primary structure (see Supporting Information Figure
S1A).1ꢀ4 Among NPP family members, NPP1 regulates the
extracellular phosphate levels by hydrolyzing nucleotide sub-
strates, such as ATP, thereby modulating bone formation, insulin
receptor signaling, and type 2 diabetes.5ꢀ13 NPP2/Autotaxin is
identical to lysophospholipase D (lysoPLD) and has key roles in
various pathophysiological processes such as angiogenesis, de-
velopment of neuropathic pain, lymphocyte trafficking, and
tumor cell invasion.14ꢀ19 Fluorescence probes for NPP2, such
Received: February 9, 2011
Published: July 01, 2011
r
2011 American Chemical Society
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TG is an excellent fluorophore which has favorable chemical
properties, including high water solubility and strong fluore-
scence. Further, the fluorescence properties of TG can be easily
controlled by the photoinduced electron transfer (PeT)
mechanism.37 Therefore, TG is widely used as a scaffold for
off/on type fluorescence probes, as in TG-βgal and TG-Phos for
detection of β-galactosidase and alkaline phosphatase (ALP),
respectively.38,39
Strategy To Detect NPP6 Activity. Our strategy to selectively
detect NPP6 activity is shown in Figure 1. Our probe, TG-mPC,
is almost nonfluorescent owing to the operation of the acceptor-
PeT (a-PeT) mechanism.37 However, 2-Me-4-OMe TG, a
possible product of lysoPLC/NPP6 reaction, is fully fluorescent.
In contrast, TG-mPhos, a possible product of lysoPLD reaction
mediated by NPP2/ATX, is almost nonfluorescent. It is possible
that TG-mPC would be hydrolyzed by NPP2 (cleavage at the
position indicated by a shadowed arrow in Figure 1), generating
TG-mPhos, but because TG-mPhos is almost nonfluorescent, we
speculated that TG-mPC would be able to selectively detect
NPP6 activity as a fluorescence enhancement.
Synthesis of TG-mPC. We synthesized TG-mPC in five steps
(Scheme 1). Compound 2 was synthesized according to the
reported method.40 This structure was an intermediate in the
synthesis of a water-soluble prodrug with a phosphate group.41,42
Compound 2 was considered useful because it has high reactivity
toward phenolic groups and is stable.
Characteristics of TG-mPC. Photochemical properties of TG-
mPhos, TG-mPC, and 2-Me-4-OMe TG are summarized in
Table 1. The fluorescence quantum yield (ΦFl) of TG-mPC took
a quite low value (ΦFl = 0.009), whereas that of 2-Me-4-OMe
TG was high (ΦFl = 0.84). TG-mPhos, the product expected to
be generated by the reaction with NPP2, also showed a low value
(ΦFl = 0.012). Thus, it is expected that detection of NPP6
activity is possible as a result of the strong fluorescence enhance-
ment, whereas hydrolysis of TG-mPC by NPP2 would not result
in fluorescence enhancement.
Reactivity of TG-mPC with NPPs. We examined the reactivity
of TG-mPC with each NPP (NPP1ꢀ7) recombinant protein
(Figure 2A,B) and found that TG-mPC is an NPP6-selective
fluorescence probe in in vitro assay: fluorescence enhancement
(Fl3 min/Fl0 min) was observed only when TG-mPC was mixed
with NPP6. The value of fluorescence enhancement was 165-fold
for NPP6, with much lower values for other NPPs. Using
colorimetric substrates, such as pNP-TMP and pNPPC, we have
been able to discriminate various NPP activities to some extent
(see Supporting Information Figure S1B,C). It should be noted
that TG-mPC can distinguish NPP6 and NPP7, which could not
be discriminated by using colorimetric substrates. We confirmed
that the product generated by enzymatic reaction with NPP6 is
2-Me-4-OMe TG by means of HPLC (see Supporting Informa-
tion Figure S3). We also examined whether TG-mPC is hydro-
lyzed by other NPPs to form nonfluorescent TG-mPhos and
found that this was not the case (see Supporting Information
Figures S4,5). Because formaldehyde is generated as a byproduct,
we tested whether formaldehyde affects the NPP6 activity; even
at 10 μM, formaldehyde had no influence on the enzymatic
activity of NPP6 (see Supporting Information Figure S6). As
shown in Figure 2C and Figure S13 (see Supporting In-
formation), TG-mPC was not hydrolyzed by porcine liver
esterase (PLE), alkaline phosphatase (ALP), or acetylcholines-
terase (AchE), further confirming its selectivity for NPP6.
Figure 1. Strategy to selectively detect NPP6 activity. TG-mPC con-
sists of three moieties: NPP6 recognition moiety, linker moiety, and
fluorophore. TG-mPC is almost nonfluorescent before enzymatic reac-
tion. NPP6 (cleavage site shown by black arrow) generates strongly
fluorescent 2-Me-4-OMe TG with elimination of phosphorylcholine
and formaldehyde (upper scheme). On the other hand, NPP2 (cleavage
site shown by shadowed arrow) generates almost nonfluorescent TG-
mPhos with the elimination of a choline (bottom scheme).
(PC-NAE) in brain.31 Highly sensitive activity sensors and
potent inhibitors of NPP6 would be useful to establish the
function of NPP6 in more detail. In addition, many kinds of
high-throughput screening (HTS) systems employing fluores-
cence probes have been used to discover small-molecular
inhibitors of a variety of targets. This methodology is extremely
useful for the identification of novel inhibitors from large
chemical libraries, not only as candidate therapeutic agents
but also as chemical tools for functional studies of the targets,
because of the high sensitivity of fluorescence-based HTS,
compared with classical absorption-based assay.32 For exam-
ple, a recently developed fluorescence probe for NPP2/ATX,
CPF4, was used to discover NPP2/ATX inhibitors20 and was
also employed in biochemical characterization of ATX.21 So, in
this study, we report the development of a novel NPP6-
selective fluorescence probe, TG-mPC, and the discovery of
a potent and selective NPP6 inhibitor by means of screening
with the use of this probe. Although many fluorescence probes
targeting phospholipases have been developed, no previously
reported probe can distinguish between the enzymatic activ-
ities of lysoPLC and lysoPLD.22,33,34 Herein, we have em-
ployed a novel design strategy to obtain TG-mPC, which
can clearly distinguish between the activities of lysoPLC and
lysoPLD.
’ RESULTS AND DISCUSSION
Design of NPP6-Selective Probe. We designed 2-Me-4-OMe
TokyoGreen-methyleneoxy phosphorylcholine (TG-mPC) as
an NPP6-selective probe (Figure 1). TG-mPC consists of three
moieties; an NPP6 recognition moiety, a linker moiety, and a
fluorophore moiety. Phosphorylcholine was selected as the
NPP6 recognition moiety, and oxymethylene as the linker
moiety, which serves to reduce steric hindrance between the
fluorophore and NPP6, and to enhance the stability of 2-Me-4-
OMe TokyoGreen-methyleneoxy phosphate (TG-mPhos) and
TG-mPC.35,36 The oxymethylene linker is eliminated as formal-
dehyde during the hydroxylation of phosphorylcholine by NPP6.
As the fluorophore moiety, we selected TokyoGreen (TG).37
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Scheme 1. Synthesis of TG-mPCa
a Reagents and conditions: (a) chloromethyl chlorosulfate, n-Bu4NHSO4, NaHCO3, CH2Cl2/H2O, 0 ꢀC to rt, 67%; (b) 2, Cs2CO3, DMF, rt, 43%; (c)
Pd/C, MeOH/CH2Cl2, rt; (d) chloranil, CH2Cl2/H2O, rt, 24% in two steps; (e) bromocholine bromide, DIEA, DMF, 50 ꢀC, 17%.
TG-mPC Analogues: Reactivity and Specificity. We synthe-
sized various TG-mPC analogues and examined their reactivity
and specificity toward various NPPs, including NPP6 (Figure 3
and see Supporting Information Scheme S1). As shown in
Figure 3 and Figure S7 (see Supporting Information), at the
concentration of 1 μM, NPP6 hydrolyzed TG-mPC, TG-
mPC3C, TG-mPC5C, TG-mPENE, and TG-mPEA. The rank
order is TG-mPC > TG-mPENE = TG-mPC3C > TG-mPC5C =
TG-mPEA. NPP2, on the other hand, showed low activity
toward these substrates; NPP2 weakly hydrolyzed TG-mPEA,
TG-mPhos, TG-mPA, TG-mPMe, and TG-mPPr, affording a
modest fluorescence increment. The TG-mPC analogues did
not show any fluorescent increment with the other NPPs, i.e.,
NPP1, -3, -4, -5, and -7. We further examined the specificity of
TG-mPC analogues for NPP6 and NPP2 using different con-
centrations of each substrate. The analyses revealed that TG-
mPC, TG-mPC3C, and TG-mPENE are highly selective probes
for NPP6 over NPP2. TG-mPC3C and TG-mPC5C have a
trimethylammonium group but have different linker lengths
from TG-mPC, while TG-mPENE and TG-mPEA have differ-
ent substituents of the amino group from TG-mPC (cationic
moiety). TG-mPhos and TG-mPA have anionic structures
(anionic moiety), and TG-mPMe and TG-mPPr have alkyl
chains (neutral moiety). These results indicate that N-substit-
uents of ethylamine (especially, quaternary > tertiary > primary
amine) are important for selective reactivity with NPP6. In
addition, the results suggest that the linker length between
phosphate group and the trimethylammonium group is critical
to the affinity for NPP6. These results support biochemical
Table 1. Photochemical Properties of TG-mPhos, TG-mPC,
TG-Phos, and 2-Me-4-OMe TGa
absorption
maximum
(nm)
extinction
coefficient
(ꢁ 104 Mꢀ1 cmꢀ1
emission fluorescence
maximum quantum
compound
TG-mPhos
)
(nm)
yield (ΦFl)
454
451
492
1.6
1.7
8.5
511
511
511
0.012
0.009
0.84
TG-mPC
2-Me-4-OMe TG
a Data were measured in sodium phosphate buffer (pH 7.4). For
determination of the fluorescence quantum yield, fluorescein in aq 0.1 N
NaOH (ΦFl = 0.85) was used as a fluorescence standard.
indications that NPP6 strongly requires a choline partial
structure in its substrates.3 TG-mPMe and TG-mPPr had
higher reactivity with NPP2 than NPP6, so that TG-mPMe
and TG-mPPr may be applicable to NPP2 activity assay.
Kinetic Study of TG-mPC and TG-mPC3C. Next, we deter-
mined the kinetic parameters of TG-mPC and TG-mPC3C.
The Km and kcat values for TG-mPC and known substrates are
shown in Table 2. The Km and kcat values of TG-mPC for NPP6
were 8.7 μM and 28 sꢀ1, respectively, and those of p-nitro-
phenylphosphorylcholine (pNPPC), a classical NPP6 sub-
strate, were 11.5 μM and 14 sꢀ1. This result indicates that
TG-mPC has approximately the same reactivity as pNPPC,
while TG-mPC3C has a lower affinity for NPP6 (Km = 32.3 μM)
than does TG-mPC. Moreover, TG-mPC has a much lower
affinity for NPP2 than does CPF4, a classical NPP2 fluorescent
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Figure 2. Reactivity of TG-mPC with NPPs and other hydrolases. (A) Time course of enzymatic reaction of TG-mPC (1 μM) with various
recombinant NPPs. NPPs were added at 120 s. Reactions were performed in NPP buffer (100 mM Tris-HCl (pH 9.0), containing 500 mM NaCl, 5 mM
MgCl2, and 0.05% Triton-X100) at 37 ꢀC. Bold black and deep gray lines indicate NPP6 and NPP2, respectively. Excitation and emission wavelengths
were 490 and 510 nm, respectively. (B) Fluorescence enhancement (Fl 3 min/Fl 0 min) was calculated from the result in A. The reaction up to 3 min was
linear. (C) Cross-reactivity of TG-mPC (1 μM) with porcine liver esterase (PLE) 5 units/mL or alkaline phosphatase (ALP). ALP or PLE was added at
120 and 180 s, respectively.
Table 2. Km and kcat values of TG-mPC, TG-mPC3C, and
pNPPC toward NPP6 and Those of TG-mPC and CPF4
toward NPP2 (for details, see Supporting Information
Figure S6)
NPP6
NPP2
TG-mPC TG-mPC3C pNPPC TG-mPC CPF4
K
m (μM)
8.7
28
32.3
57
11.5
14
>400a
∼7.3
45.0
14
kcat (secꢀ1
)
a This value was too large to calculate. Details are shown in Figure S8
(see Supporting Information).
mPC and TG-mPPr, and fluorescence images were captured
for 3 min after adding each probe. As shown in Figure 4C, the
fluorescence intensities of NPP6 (+) HeLa cells and the
surrounding medium were increased with TG-mPC, while
there was no increase in the case of NPP6 (ꢀ) cells or when
TG-mPPr was used in NPP6 (+) cells. The fluorescence of
NPP6 (+) cells was significantly increased compared with
NPP6 (ꢀ) cells (Figure 4D). These results indicate that TG-
mPC is suitable for cell-based assay.
Screening of NPP6 Inhibitors and Evaluation. Using TG-
mPC as a substrate, we next performed NPP6 inhibitor screening
of a chemical library containing about 80 000 compounds. The
initial screening identified 19 compounds showing inhibitory
activity (>50% at 10 μM concentration). The compounds were
further tested in a second cycle of NPP6 assay, and 16 out of 19
were confirmed to inhibit NPP6 with IC50 vales ranging from
2.1 μM (TPC-010) to 16.7 μM (TPC-024) (Table 3, see Supporting
Information Figures S10ꢀS12). TPC-010 and TPC-011 showed
similar inhibitory activity (IC50 = 2.1, 4.4 μM, respectively,
Figure 5A,B) for NPP6, but they were not specific to NPP6
(Figure 5C). Among these compounds, we focused on TPC-
009 because it efficiently (IC50 = 2.7 μM) and selectively
inhibited NPP6 over other NPP family members (Figure 5C).
In addition, TPC-009 has a suitable structure for chemical mod-
ification and is water-soluble.
Figure 3. Reactivity and specificity of TG-mPC analogues toward
NPP6. (A) Structures of TG-mPC analogues, TG-mPC3C, TG-mPC5C,
TG-mPENE, TG-mPEA, TG-mPA, TG-mPMe, and TG-mPPr. (B)
Reactivity of TG-mPC analogues with recombinant NPP6. The values of
fluorescence enhancement (Fl3 min/Fl0 min at 510 nm after the addition
of NPPs) are shown. For details, see Supporting Information Figure S7.
(C) Specificity for NPP6 against NPP2. The values of fluorescence
enhancement of NPP6/NPP2 (FI3 min/0 min (NPP6)/FI27 min/0 min
(NPP2) at 510 nm) are shown. Inset: specificity for NPP2 against NPP6.
substrate (see Supporting Information Figure S8). These
results clearly demonstrate that TG-mPC is a selective sub-
strate for NPP6.
Cell-Based Application. We tested the suitability of TG-
mPC for cell-based assay. First, we incubated TG-mPC in cell
lysates prepared from NIH3T3 cells expressing NPP6 and
from control NIH3T3 cells. TG-mPC showed a fluorescence
increment in NPP6 (+) cell lysate but not in NPP6 (ꢀ) cell
lysate (Figure 4A). We also applied TG-mPC to the cell
cultures. Significant fluorescent increment was observed when
TG-mPC was added to media of NPP6-expressing NIH3T3
cells but not control NIH3T3 cells (Figure 4B). These results
indicate that TG-mPC is hydrolyzed only by NPP6 among the
cellular enzymes. Further, we examined whether or not TG-
mPC is suitable to visualize NPP6 activity at the cellular level.
HeLa cells expressing NPP6 were incubated with 10 μM TG-
StructureꢀActivity Relationship: Amine Substituents and
Linker Length. To obtain more potent NPP6 inhibitors, we
performed a structureꢀactivity relationship (SAR) study of
TPC-009. For this purpose, we focused on several compounds
with similar structure to TPC-009, which had been included in
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Figure 4. Cell-based assay with TG-mPC. (A) Cell lysate assay with TG-mPC. Cell lysate; NPP6 (+) NIH3T3 cell lysate, NPP6 (ꢀ) NIH3T3 cell
lysate. The preparation of each lysate is described in Experimental Section. (B) Total NPP6 activity, i.e., membrane-associated and secreted NPP6
activity, of NPP6 (+) NIH3T3 cells and NPP6 (ꢀ) NIH3T3 cells. Excitation wavelength was 490 nm. (C) Fluorescence microscopic imaging of NPP6
activity in living cells with TG-mPC and TG-mPPr. HeLa cells expressing NPP6 or mock with mCherry were incubated with 10 μM solution of each
probe in Hank’s Balanced Salt Solution (HBSS) at 37 ꢀC. Confocal fluorescence images and DIC images were captured at 3 min after addition of each
probe. Arrowheads indicate NPP6 (+) HeLa cells. (D) Comparison of fluorescence intensities between NPP6 (+) and NPP6 (ꢀ) HeLa cells after
addition of TG-mPC (final 10 μM) and TG-mPPr (final 10 μM). The results are mean ( SD.
Table 3. Inhibition (%) of NPP6 by Screening-Identified
Compounds (TPC-009 to -011, and TPC-014 to -029, >50%
inhibition at 10 μM) and the IC50 Valuesc
the initial screening but had shown little or no inhibitory
activity. Ten such compounds, named T1ꢀT10 (Figure 6A),
all had an isothiourea moiety as a partial structure. Among these
compounds, T5 and T7 but not T1 inhibited NPP6. The rank
order was T5 > T7 .T1 (Figure 6B). Thus, the linker length
and the substituents of the amine moiety seem to be critical for
the inhibitory activity. We therefore synthesized T11, which has
a trimethylammonium group instead of the dimethylamino
group in TPC-009 (see Supporting Information Scheme S2).
T11 was found to be a potent and selective NPP6 inhibitor with
an IC50 value of 0.21 μM (Figure 7A,B). Kinetic analysis
revealed that T11 inhibits NPP6 in a competitive manner
(Figure 7C). We also examined the effects of linker length
and substituents of the amine moiety (primary amines, tertiary
amines, and quaternary amines) on the inhibitory activity
(Figure 8A,B). As a result, the length of linker, C3, was found
to be optimum. These experiments identified T11 as a potent
NPP6 inhibitor with an optimum C3 linker between the amine
moiety and isothiourea moiety, and a favorable cationic
trimethylammonium group.
StructureꢀActivity Relationship: Isothiourea Substituents.
We also examined the substituents of isothiourea. The compounds
we synthesized are shown in Figure 9A, and their IC50 values are
summarized in Figure 9B. Isothiourea monosubstituted with mod-
erate-sized substituents (methyl (T19), benzene (T16), and bis-
(trifluoro)benzene (T17), but not naphthalene (T18)) showed
inhibitory activity nearly identical to that of T11. On the other hand,
disubstituted isothiourea compounds, such as dimethyl (T20),
diethyl (T21), dibutyl (T22), cyEt (T23), and cyPr (T24), showed
inhibitory activity lower than that of S11. However, trisubstituted
inhibition (%, at
compound
IC50 (μM)
10 μM in the screening)
TPC-009
TPC-010
TPC-011
TPC-014
TPC-015
TPC-016
TPC-017
TPC-018
TPC-019
TPC-020
TPC-021
TPC-022
TPC-023
TPC-024
TPC-025
TPC-026
TPC-027
TPC-028
TPC-029
2.7
2.1
4.4
6.7
4.3
96
92
62
54
76
62
64
78
55
71
66
74
53
50
60
62
55
88
55
a
ꢀ
11.1
5.5
a
ꢀ
5.6
5.0
9.8
4.5
16.7
11.1
5.9
16.2
3.9
b
ꢀ
a No inhibitory activity (IC50 > 50 μM). b This compound shows
fluorescence that interfered with the measurement of its IC50, but it
has inhibitory activity toward NPP6. c For the structures of TPC-014 to
-029, see Supporting Information Figure S10.
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Figure 5. Inhibitory activity and selectivity of TPC-009, -010, -011 toward NPP6. (A) Structures of TPC-009, -010, -011, hit compounds in the primary
screening. (B) Inhibition plots and curves of TPC-009, -010, -011. The IC50 values were 2.7, 2.1, 4.4 μM, respectively. The results are mean ( SD (n = 6).
(C) Selectivity test of TPC-009, -010, -011 (50 μM) toward recombinant NPPs. The results are mean ( SD (n = 3). These values were determined by
using absorption-based substrates, pNP-TMP (for NPP1ꢀ5) and pNPPC (for NPP6, -7) at 405 nm.
Figure 6. Inhibitory activity of primary screening samples toward NPP6. (A) Structures of TPC-009 and its analogues (T1ꢀ10) which showed low
inhibitory activity in the primary screening. Inhibition (%) by the indicated compounds at 10 μM is shown in brackets. The inhibition (%) of T1, T2, T5,
and T7 was measured twice. (B) Inhibition plots and curves of T1, T5, and T7 on the retest. IC50 values were >50 μM (N.D. = not determined), 5.3 μM,
and 29.5 μM, respectively. The results are mean ( SD (n = 6).
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Figure 7. Inhibition plots and inhibition curve of T11 toward NPP6. (A) The IC50 value of T11 was 0.21 μM. The results are mean ( SD (n = 6). Inset:
the structure of T11. (B) Selectivity test of T11 (50 μM) toward recombinant NPPs. The results are mean ( SD (n = 3). (C) LineweaverꢀBurk plots of
NPP6 inhibition, showing competitive inhibition by T11.
isothiourea (trimethyl, T25) showed strong inhibitory activity, like
that of T11.
’ CONCLUSION
The biological functions of NPP4ꢀ7 have not been elucidated
in detail, and this fact led us to design and synthesize novel
fluorescence probes for NPP6, i.e., TG-mPC and its derivatives,
which were rationally designed as off/on type fluorescence probes
based on the PeT mechanism. We anticipate that TG-mPC will be
far more useful than previously reported NPP6 substrates, such as
glycerophosphocholine (GPC), lysophosphatidylcholine (LPC),
and p-nitrophenylphosphorylcholine (pNPPC), because it can
detect NPP6 activity with high sensitivity and distinguish the
activity of NPP6 from those of other NPP family members,
including NPP2/autotaxin, as a consequence of our design
strategy (for details, see Supporting Information Figure S1B,C
and Figure 1). Previously reported probes, such as DDPB, could
not distinguish between phospholipase D (PLD) and phospholi-
Figure 8. T11 derivatives with various substituted amines and linker
length (T12ꢀ15 and compounds in the primary screening). (A)
Structures of T12ꢀ15. (B) IC50 values (μM) of the indicated com-
pounds are shown. Synthesis of these compounds is shown in Scheme S3
(see Supporting Information).
pase C (PLC) activities.33 Thus, TG-mPC is a novel, highly
selective probe for lysoPLC (NPP6). Our strategy, as shown in
Figure 1, in fact proved unnecessary, because it turned out that
NPP2 did not hydrolyze TG-mPC, but nevertheless, this approach
might be applicable to other enzymatic reactions, especially other
phosphodiesterases that distinguish different cleavage sites.
Study of the reactivity of TG-mPC analogues with NPPs and
the SAR of NPP6 inhibitors may also give us insight into the
structure of NPP6. The recently solved NPP2 structure suggests
that the difference of TG-mPC reactivity between NPP2 and
NPP6 derives from the insertion loop, which is conserved in NPP
family members except NPP2, in the proximity of the catalytic
center.43,44 Although the catalytic machinery is well conserved
among the NPP family members, and they have a common native
substrate, LPC, the substrate enters the catalytic pocket in
opposite orientations in NPP2 and NPP6. Namely, the NPP2
catalytic pocket recognizes the fatty acid of LPC, while the NPP6
catalytic pocket recognizes the phosphorylcholine residue, and
this determines the phosphodiester bond to be cleaved (lysoPLC
vs lysoPLD). The phosphorylcholine residue in TG-mPC and its
structural analogues are recognized by NPP6, and this may be the
reason why they are NPP6-specific. It is noteworthy that TG-
mPC discriminate between NPP6 and NPP7. Because previously
reported colorimetric substrates do not discriminate these two
Figure 9. T11 derivatives with substituted isothiourea (T16ꢀ25). (A)
Structures of T16ꢀ25. T16ꢀS19 are monosubstituted isothioureas,
T20ꢀ24 are disubstituted isothioureas, and T25 is a trisubstituted
isothiourea. (B) IC50 values of T11 derivatives with substituted iso-
thioureas are shown. Synthesis of these compounds is shown in Scheme
S4 (see Supporting Information).
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NPPs, TG-mPC appears to have another moiety that is specifi-
cally recognized by NPP6.
(s = singlet, d = doublet, t = triplet, br = broad, and m = multiplet),
integration, and J coupling. Mass spectra were recorded on a JEOL JMS-
T100LC mass spectrometer. Silica gel column chromatography was
performed using silica gel 60 (Kanto Chemical Co., Inc., Japan) and
Chromatorex-NH silica gel (Fuji Silysia Chemical, Kasugai, Japan). Pre-
parative HPLC purification was performed on a reverse-phase ODS
column (GL Sciences, Japan, Inertsil ODS-3 10 mm ꢁ 250 mm) using
0.1% trifluoroacetic acid in water as eluent A and 0.1% trifluoroacetic acid,
80% acetonitrile, and 20% water as eluent B, or 100 mM TEAA in water as
eluent A and 80% acetonitrile, and 20% water as eluent B, with a JASCO
PU-2080 plus system, at a flow rate of 5.0 mL/min. HPLC analyses were
performed on a reversed-phase ODS column (GL Sciences, Inertsil ODS-3
4.6 mm ꢁ 250 mm), fitted on a JASCO PU-980 plus system, at a flow rate
of 1.0 mL/min. UVꢀvisible spectra were obtained on a Shimadzu UV-
1600 (Tokyo, Japan). Fluorescence spectra were obtained on a Hitachi
F4500 (Tokyo, Japan). Fluorescence assay on 384-well plates were
performed using Perkin-Elmer EnVision Multilabel Plate Readers
(Beaconsfield, Buckinghamshire, England) or PHERAstar (BMG LAB-
TECH, Germany)
Determination of Kinetic Parameters. Various concentrations
of probes (TG-mPC, TG-mPC3C, pNPPC, and CPF4) were dissolved
in NPP buffer (100 mM Tris-HCl buffer (pH 9.0), containing 500 mM
NaCl, 5 mM MgCl2, and 0.05% Triton-X100). NPP6 or NPP2 was
added to the solution, and the fluorescence intensity was recorded
continuously. Initial reaction velocity was calculated and plotted against
probe concentration, and the points were fitted to a MichaelisꢀMenten
curve. Km and Vmax were determined by the least-squares method.
HPLC Analysis. To confirm that the enzymatic reaction took place,
reversed-phase HPLC analysis was performed using an Inertsil 3 ODS
column (4.6 mm ꢁ 250 mm, GL Science, Japan), fitted on a JASCO
PU980 HPLC system. Conditions and recorded absorption wavelength
are given in the figure legends.
Fluorometric Analysis. The slit width was 2.5 nm for both
excitation and emission. The photomultiplier voltage was 750 V. TG-
Phos, TG-mPC, TG-mPhos, and TG-mPC analogues were dissolved in
NPP buffer to obtain 500 μM stock solutions. Relative fluorescence
quantum yields (ΦFl) were obtained by comparing the area under the
emission spectrum of the probes with that of a solution of fluorescein
(ΦFl = 0.85) in aq 0.1 N NaOH.
ALP, PLE, and AchE Assay. ALP assay was performed in ALP
buffer (50 mM Tris-HCl buffer (pH 8.0), containing 0.5 mM MgCl2).
PLE assay was performed in 100 mM sodium phosphate buffer (pH 7.4)
at room temperature. AchE assay was performed in phosphate-buffered
saline (PBS, pH 7.4) at room temperature.
NPP6 Inhibitor Screen. High-throughput screening (HTS) was
performedagainst apart ofthechemicallibrary(80 000compounds) ofthe
Chemical Biology Research Initiative, The University of Tokyo. Screening
methods were as follows; at first, small molecules (2 mM DMSO stock
solution) from library plates (320 compounds/384 wells) were diluted
with NPP buffer to 30 μM solution (1.5% DMSO) by the Multi-Dispensor
EDR 384 (BioTec, Japan). Next, TG-mPC (1.5 μM, no DMSO) solution
(5 μL) was dispensed into the individual wells of the assay plates, and the
prepared solution (5 μL) of compounds was also added into each well.
Finally, NPP6 solution (5 μL) was dispensed into the plates (total volume
was 15 μL), and the plates were incubatedat room temperature for 60 min.
TG-mPC is also applicable to cell-based assay without inter-
ference from other enzymes, such as esterases and phosphatases
(Figure 2C and see Supporting Information Figure S13). It was
confirmed to be useful for cell-based NPP6 inhibitor screening and
is expected to be suitable for real-time imaging of NPP6 activity in
NPP6-expressing cells such as oligodendrocytes and kidney
epithelial cells.45 These projects are ongoing in our laboratory.
We found a potent and selective NPP6 inhibitor, T11
(Figures 5ꢀ9). Structureꢀactivity relationship (SAR) analyses
revealed that the structure of T11, which consists of trimethylam-
monium, propyl linker, and isothiourea, is optimum among the
compounds synthesized in this study. We also examined the binding
affinity of T11 for histamine receptors (H1ꢀH4), inducible NOS
(iNOS), and constitutive NOS (cNOS), and the inhibitory activity
toward acetylcholinesterase (AchE) (see Supporting Information
Figure S14). T11 showed inhibitory activity toward H4 receptor at
10 μM (about 50% inhibition) but had no effect on the other
enzymes. Our proposed inhibition mechanism of T11 is shown in
Figure S15 (see Supporting Information). T11 has a similar
structure to LPC, i.e., a natural substrate of NPP6. The trimethyl-
ammonium group is common, and a chelating moiety for Zn2+ is
seen in both structures: phosphate group for LPC and isothiourea
moiety for T11. In this report, we especially focused on the structure
of TPC-009; however, other compounds may be still valuable as
lead compounds of highly potent NPP6-selective inhibitors. For
instance, it is considered that TPC-011, which has a scaffold different
from that of TPC-009, may be an attractive candidate as an NPP6
inhibitor, because it has comparatively potent inhibitory activity and
high selectivity for NPP6. Moreover, we have already examined the
SAR of TPC-011 (see Supporting Information Figure S16).
Although the results are not entirely clear-cut, TPC-011 derivatives
may prove to be potent and selective NPP6 inhibitors.
In summary, we have developed a novel fluorescence probe,
TG-mPC, which can selectively detect NPP6/lysophospholipase
C activity, and especially, distinguish NPP6 from NPP2/lysopho-
spholipase D based on our rational design strategy. We con-
firmed that it can be utilized in cellular systems. We think that
TG-mPC will be useful to elucidate the function of NPP6,
because it is the first NPP6-selective probe. We also found a
novel and potent NPP6-selective inhibitor, T11. We believe that
T11 cannot only contribute to the elucidation of NPP6 function
as an alternative to knockout mice or RNAi but also may have
wider application, e.g., in the development of candidate ther-
apeutic agents for treatment of NPP6-related diseases, because it
is the first-in-class NPP6 inhibitor.46ꢀ49
’ EXPERIMENTAL SECTION
Materials. ALP was purchased from Sigma-Aldrich Japan. p-Nitro-
phenylthymidine 50-monophosphate (pNP-TMP) and p-nitrophenylpho-
sphorylcholine (pNPPC) were purchased from Sigma. All other reagents
and solvents listed below or described in the legend to each synthetic
scheme were purchased from Tokyo Kasei Co., Ltd. (Japan), Wako Pure
Chemical Industries, Ltd. (Japan), or Aldrich Chemical Co. Dehydrated
DMF, methanol, ethanol, dichloromethane, and pyridine were 99% purity
or greater. Unless otherwise specified, reagents were of the lowest
The fluorescence of 2-Me-4-OMe TG was measured (λex = 490 nm, λem
=
510 nm) for all plates by a microplate reader. Background control wells
(i.e., no enzyme wells, n = 4) were also prepared for all plates.
Analysis of the Inhibitor Screening. Inhibition (%) of all
samples was calculated by use of the following eq A.
1
acceptable grade and were used without further purification. H NMR
spectra were recorded on JEOL JNM-LA300 and JEOL JNM-LA400
instruments; δ values are given in ppm from the solvent resonance as the
internal standard. Data are reported as follows: chemical shift, multiplicity
inhibition ð%Þ ¼ 100 ꢁ ½ðFIsample ꢀ FIbackgroundÞ=ðFImedian ꢀ FIbackgroundÞꢂ
ðAÞ
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ARTICLE
where FIsample is the fluorescence intensity of the sample, FIbackground is
the average fluorescence intensity of four background control samples,
and FImedian is the average fluorescence intensity of two median samples
from each plate (320 samples).
’ ACKNOWLEDGMENT
This work was supported in part by the Ministry of Education,
Culture, Sports, Science and Technology of Japan (grant nos.
22000006 to T.N., 20689001 and 21659024 to K.H., and
21750135 to T.T.), and by a grant from the Industrial Technol-
ogy Development Organization (NEDO) of Japan (to T.T.).
K.H. was also supported by Sankyo Foundation of Life Science,
and M.K. was supported by a Grant-in-Aid for JSPS Fellows. This
work was supported in part by funds from the Target Protein
Research Programs from the Ministry of Education, Culture,
Sports, Science and Technology of Japan.
Selectivity Test. Inhibitory activity of the hit compounds toward
NPP1ꢀ7 was calculated using pNP-TMP (for NPP1ꢀ5) and pNPPC
(for NPP6, -7). The final concentration of these two absorption-based
substrates was 100 μM. Assay protocol was as follows: assay buffer (NPP
buffer, 37 μL) was dispensed into a 96-well microplate and 500 μM
pNP-TMP or pNPPC (20 μL) was dispensed into the microplate. Then,
30 μM samples (33 μL) were added, and finally NPP1ꢀ7 solution (10 μL)
was added to each well. Total volume of each well was 100 μL. The
plate was incubated for 2 h, and absorbance (405 nm) was measured
by a microplate reader.
Preparation of Recombinant NPPs. Recombinant NPPs were
prepared as follows. HEK293 cells were cultured in Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with antibiotics, glutamine,
and 10% fetal bovine serum under an atmosphere of 5% CO2 at 37 ꢀC.
HEK293 cells were transfected with the cDNAs using Lipofectamine
2000 reagent (Invitrogen) according to the manufacturer’s protocol.
Cell culture media were collected 48 h after the transfection. Collected
culture media of all NPPs were used without further purification.
Cell-Based Assays. NPP6-expressing NIH3T3 cells and nonex-
pressing NIH3T3 cells were cultured in DMEM supplemented with
penicillin (100 units/mL), streptomycin (100 μg/mL), glutamine, and
10% fetal bovine serum in a humidified incubator containing 5% CO2
gas at 37 ꢀC. Lysates of NPP6-expressing NIH3T3 cells and nonexpres-
sing cells were prepared with CelLyticTMM (Sigma-Aldrich) according
to the manufacturer’s protocol.
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’ ASSOCIATED CONTENT
S
Supporting Information. Synthesis description, sup-
b
porting figures, and complete references. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
tlong@mol.f.u-tokyo.ac.jp
Notes
†Abbreviationsused:NPP, nucleotidepyrophosphatase/phospho-
diesterase; lysoPLC, lysophosholipase C; lysoPLD, lysopho-
sholipase D; LPC, lysophoshitidylcholine; SM, sphingomyelin;
GPC, glycerophoshorylcholine; pNP-TMP, p-nitrophenylthy-
midine 50-monophosphate; pNPPC, p-nitrophenylthymidine-
phoshorylcholine; FAAH, fatty acid amide hydrolase; ALP,
alkaline phosphatase; PLE, porcine liver esterase; AChE,
acetylcholine esterase; NOS, nitric oxide synthase; TG, To-
kyoGreen; TG-mPC, methylenoxyphosphorylcholine; PeT,
photoinduced electron transfer; HTS, high-throughput
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