Fluorogenic phospholipid substrate to detect lysophospholipase D/autotaxin activity

Article abstract of DOI:10.1021/ol060414i

Lysophospholipase D (lysoPLD), also known as autotaxin (ATX), is an important source of the potent mitogen lysophosphatidic acid (LPA). Two fluorogenic substrate analogues for lysoPLD were synthesized in nine steps from (S)-PMB-glycerol. The substrates (FS-2 and FS-3) show significant increases in fluorescence when treated with recombinant ATX and have potential applications in screening for this emerging drug target.

Full text of DOI:10.1021/ol060414i

ORGANIC
LETTERS
2006
Vol. 8, No. 10
2023-2026
Fluorogenic Phospholipid Substrate to
Detect Lysophospholipase D/Autotaxin
Activity
Colin G. Ferguson,*,† Cleve S. Bigman,† Robyn D. Richardson,†
Laurens A. van Meeteren,‡ Wouter H. Moolenaar,‡ and Glenn D. Prestwich§
Echelon Biosciences Inc., 675 Arapeen DriVe 302, Salt Lake City, Utah 84108,
The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam,
The Netherlands, and Department of Medicinal Chemistry, UniVersity of Utah,
Salt Lake City, Utah 84108
cferguson@echelon-inc.com
Received February 16, 2006
ABSTRACT
Lysophospholipase D (lysoPLD), also known as autotaxin (ATX), is an important source of the potent mitogen lysophosphatidic acid (LPA).
Two fluorogenic substrate analogues for lysoPLD were synthesized in nine steps from (S)-PMB-glycerol. The substrates (FS-2 and FS-3) show
significant increases in fluorescence when treated with recombinant ATX and have potential applications in screening for this emerging drug
target.
Bioactive lysophospholipids, such as lysophosphatidic acid
(LPA) and sphingosine 1-phosphate (S1P), exhibit pleio-
morphic effects on multiple cell lineages, including ovarian
cancer cells.1 LPA and S1P signal through specific cell
surface receptors of the endothelial cell differentiation gene
(formerly known as edg) family of cell surface seven-
transmembrane-domain G-protein-coupled receptors.2 The
purification, characterization, and identification of the ovarian
cancer activating factor (OCAF) from ascites of ovarian
cancer patients demonstrated that OCAF is comprised of
numerous forms of LPA and accounts for the ability of
ascites to activate ovarian cancer cells.3 LPA, at concentra-
tions present in ascites from ovarian cancer patients (1-80
µM), increases proliferation under anchorage-dependent and
-independent conditions, prevents apoptosis and anoikis,
increases invasiveness, induces cytoskeletal reorganization
and change of shape, and decreases sensitivity to cisplatin.4
LPA can arise through at least two routes: the loss of the
sn-2 acyl chain by phosphatidic acid-specific PLA2 or
cleavage of the choline group of lysophosphatidylcholine
(LPC) by lysophospholipase D (lysoPLD) (Figure 1A).
LysoPLD activity was first characterized over 16 years ago
and has important roles in normal physiology as a source of
plasma LPA.5 Two laboratories have independently deter-
† Echelon Biosciences Inc.
(3) Mills, G. B.; May, C.; McGill, M.; Roifman, C. M.; Mellors, A.
Cancer Res. 1988, 48, 1066.
‡ Netherlands Cancer Institute.
§ University of Utah.
(4) (a) Mills, G. B.; Bast, R. C., Jr.; Srivastava, S. J. Nat. Cancer Inst.
2001, 93, 1437. (b) Mills, G. B.; Eder, A.; Fang, X.; Hasegawa, Y.; Mao,
M.; Lu, Y.; Tanyi, J.; Tabassam, F. H.; Wiener, J.; Lapushin, R.; Yu, S.;
Parrott, J. A.; Compton, T.; Tribley, W.; Fishman, D.; Stack, M. S.;
Gaudette, D.; Jaffe, R.; Furui, T.; Aoki, J.; Erikson, J. R. Cancer Treat.
Res. 2002, 107, 259. (c) Tanyi, J. L.; Morris, A. J.; Wolf, J. K.; Fang, X.;
Hasegawa, Y.; LaPushin, R.; Auersberg, N.; Sigal, Y.; Newman, R. A.;
Felix, E. A.; Atkinson, E. N.; Mills, G. B. Cancer Res. 2003, 63, 1073.
(1) (a) Fang, X.; Gaudette, D.; Furui, T.; Mao, M.; Estrella, V.; Eder,
A.; Pustilnik, T.; Sasagawa, T.; Lapushin, R.; Yu, S.; Jaffe, R. B.; Wiener,
J. R.; Erikson, J. R.; Mills, G. B. Ann. N.Y. Acad. Sci. 2000, 905, 188. (b)
Luquain, C.; Sciorra, V. A.; Morris, A. Trends Biochem. Sci. 2003, 28,
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10.1021/ol060414i CCC: $33.50
© 2006 American Chemical Society
Published on Web 04/13/2006

Figure 1. A: Hydrolysis of oleoyl LPC by lysoPLD. B: Schematic of fluorescence-dequenching lysoPLD/ATX substrate. The fluor in the
substrate is quenched through intramolecular energy transfer until it is hydrolyzed by the enzyme at which point the fluorescent product can
be observed. C: Structures of fluorogenic substrates FS-2 and FS-3.
mined purified and cloned plasma lysoPLD and showed that
there was no sequence homology to other PLD enzymes.
Instead, it was identical to secreted autotaxin (ATX), a
member of the nucleotide pyrophosphatase/phosphodiesterase
(NPP) family of ecto/exo enzymes, that stimulates tumor cell
motility and has an in vivo role in tumor progression and
invasion.6 Recent reviews suggest that inhibition of increased
lysoPLD/ATX activity by small molecules could be an
attractive new avenue for anticancer chemotherapy; however,
for that to become reality, a simple assay for high throughput
screening is needed.7
application of a doubly labeled ATX substrate, CPF4, was
published.9 CPF4 is a fluorescence resonance energy transfer
(FRET) substrate derived from bis-p-nitrophenyl phosphate,
a common colorimetric phosphodiesterase substrate, and
although it was a significant improvement over the previously
described assays, it does not share many structural features
of LPC. In this report, the synthesis and validation of two
simple fluorogenic phospholipid substrates for lysoPLD/ATX
activity are demonstrated.
The envisioned substrates employ a “fluorescence de-
quenching” motif, in which a fluorophore that is “silent”
because of intramolecular FRET to a nonfluorescing quench-
er becomes fluorescent once enzymatic hydrolysis cleaves
the substrate (Figure 1B). This FRET paradigm has been
applied to fluorogenic assays for ceramidase,10 DNA ligase,11
PLA2,12 and nucleic acid hybridization.13 Because ATX is
reported to hydrolyze a variety of acyl chain modifications
and is also tolerant to changes in the backbone and
headgroup,14 a labeled ethanolamido headgroup was chosen
for FS-2 and FS-3 (Figure 1C), rather than a synthetically
more complex choline analogue. In addition, a PEG tether
was appended to the headgroup to move the bulkier fluor or
A number of different methods have been employed to
assay lysoPLD activity.8 Early work was performed with 14C-
palmitoyl-LPC and radio-thin-layer chromatography (TLC).
This was supplanted by a TLC purification of unlabeled LPA
with GC analysis of methyl esters or by measurement of
LPA-trimethylsilyl ether by gas chromatography-mass
spectrometry (GC-MS). An endpoint-type dual-enzymatic
photometric assay has recently been employed to detect
choline liberated from exogenously added LPC.6c However,
these methods are complex and are not suited to high
throughput screening. During the course of this project, the
(9) van Meeteren, L. A.; Ruurs, P.; Christodoulou, E.; Goding, J. W.;
Takakusa, H.; Kikuchi, K.; Perrakis, A.; Nagano, T.; Moolenaar, W. H. J.
Biol. Chem. 2005, 280, 21155.
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J.; Coward, J. Bioorg. Med. Chem. Lett. 2001, 11, 1561.
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Biochem. 1990, 185, 80. (b) Feng, L.; Manabe, K.; Shope, J. C.; Widmer,
S.; DeWald, D. B.; Prestwich, G. D. Chem. Biol. 2002, 9, 795. (c) Rose, T.
M.; Prestwich, G. D. ACS Chem. Biol. 2006, 1, 83.
(13) Tyagi, S.; Kramer, F. R. Nat. Biotechnol. 1996, 14, 303.
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Kishimoto, T.; Mizuno, K.; Saku, K.; Taguchi, R.; Arai, H. J. Biol. Chem.
2002, 277, 48737. (b) Clair, T.; Aoki, J.; Koh, E.; Bandle, R. W.; Nam, S.
W.; Ptaszynska, M. M.; Mills, G. B. Cancer Res. 2003, 63, 5446.
(5) Tokumura, A.; Harada, K.; Fukuzawa, K.; Tsukatani, H. Biochim.
Biophys. Acta 1986, 875, 31.
(6) (a) Tokumura, A.; Majima, E.; Kariyi, Y.; Tomingawa, K.; Kogure,
K.; Yasuda, K.; Fukuzawa, K. J. Biol. Chem. 2002, 277, 39436. (b)
Moolenaar, W. H. J. Cell Biol. 2002, 158, 197. (c) Umezo-Goto, M.; Kishi,
Y.; Taira, A.; Hama, K.; Dohmae, N.; Takio, K.; Yamori, T.; Mills, G. B.;
Inoue, K.; Aoki, J.; Arai, H. J. Cell Biol. 2002, 158, 227. (d) Morris, A. J.;
Engebrecht, J. A.; Frohman, M. A. Trends Pharmacol. Sci. 1996, 17, 182.
(7) (a) Goto, M.; Tanyi, J.; Lahad, J.; Liu, S.; Lapushin, R.; Hasegawa,
Y.; Lu, Y.; Trost, R.; Bevers, T.; Jonasch, E.; Aldape, K.; Liu, J.; James,
R. D.; Ferguson, C. G.; Xu, Y.; Prestwich, G. D.; Mills, G. B. J. Cell.
Biochem. 2004, 92, 1115. (b) Mills, G. B.; Moolenaar, W. H. Nat. ReV.
Cancer 2003, 3, 582. (c) Feng, L.; Mills, G. B.; Prestwich, G. D. Expert
Opin. Ther. Pat. 2003, 13, 1619.
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Org. Lett., Vol. 8, No. 10, 2006

Scheme 1. Synthesis of FS-2 and FS-3
quencher away from the phosphodiester and increase hy-
the end of the synthesis, resulted in poor yields. Instead, it
was protected as the tert-butyldimethylsilyl ether (5) on the
basis of examples in the literature showing TBS ethers could
be cleaved under acidic conditions. Oxidative cleavage of
the PMB group gave the chiral intermediate 6. Phosphityl-
ation of the hydroxyl with benzyl bisdiisopropylphosphor-
amidite yielded phosphoramidite 7. The protected glycerol
was converted to the phosphoramidite rather than the PEG
headgroup because previous attempts in this lab to prepare
phosphoramidites from PEG alcohols had been unsuccessful.
Coupling 2 and 7 via tetrazole proceeded smoothly followed
by oxidation with MCPBA, forming the orthogonally pro-
tected phosphate 8. Catalytic hydrogenation liberated the
PEG amine 9 which was used as a precursor for both FS-2
and FS-3. The PEG amino group was acylated via the
N-hydroxysuccinimidyl ester (SE) of dabcyl forming 10.
Treatment with TFA in CH2Cl2 removed the TBS and Boc
groups, and the resulting amine was acylated via BODIPY-
FL-SE yielding the fluorogenic substrate FS-2 (60% from
9). The route to FS-3 followed the same sequences of
reactions as FS-2. Treatment of 9 with FAM-SE gave 11
which was deprotected with TFA and labeled with dabcyl
to yield FS-3 (51% from 9).
drophilicity. Dabcyl was chosen as the quencher for both
substrates because it has been shown to effectively quench
green fluors in the previously described applications. In
FS-2, the hydrophobic fluor BODIPY-FL is appended to
the sn-1 acyl chain with dabcyl on the headgroup. For
FS-3, the positions of the quencher and fluor are reversed
and BODIPY-FL was replaced with more hydrophilic
fluorescein.
The synthesis of FS-2 and FS-3 is described in Scheme
1. A number of considerations were taken into account when
designing the synthetic strategy. Because the majority of
commercially available dyes are amine reactive, orthogonally
protected amino groups in the headgroup and monoacyl
glycerol were required, and in this case, Cbz and Boc were
chosen. In addition, a universal intermediate (9) was desired
so that different combinations of fluors and quenchers could
be potentially attached, thus allowing simple alteration of
structual and/or fluorogenic properties. The PEG headgroup
(2) was prepared by coupling commercially available Cbz-
protected PEG4 acid (1) with ethanolamine by DCC/HOBt.
To prepare the protected monoacyl glycerol segment, p-
methoxybenzyl glyceryl ether 3 (prepared from (S)-isopro-
pylideneglycerol15) was first selectively esterified at the
primary hydroxyl with N-Boc-caproic acid forming 4 in 47%
yield.16 Attempts to protect the sn-2 hydroxyl as the tert-
butyl ether, which could be removed with the Boc group at
The fluorogenic substrates were evaluated with recombi-
nant ATX. FS-2 and FS-3 showed 2.8- and 10.7-fold
increases in fluorescence, respectively, at 2.5 µM when
incubated with 75 nM ATX (Figure 2). An increase in
fluorescence was not observed when either substrate was
treated with an inactive ATX mutant (data not shown). KM
and Vmax were determined for both substrates (Table 1), but
(15) Herbert, N.; Beck, A.; Lennox, R. B.; Just, G. J. Org. Chem. 1992,
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Org. Lett., Vol. 8, No. 10, 2006
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Table 1. Michaelis-Menten Kinetics of Hydrolysis of FS-2
and FS-3 by Recombinant ATXa
b
substrate
KM (µM)
Vmax
FS-2
FS-3
3.1 ( 0.6
6.3 ( 0.2
261 ( 12.3
261 ( 2.3
a Experimental details in ref 17. b Arbitrary fluorescence in units/min.
possesses lysoPLC and sphingomyelinase activities, but there
is not enough evidence yet regarding headgroup specificity
to preclude FS-2 or FS-3 acting as substrates.20
In conclusion, two dual-labeled ATX substrate analogues
were synthesized featuring a fluor and a quencher on the
sn-1 acyl chain and the ethanolamino headgroup. Because
of the proximity of the two moieties, intramolecular energy
transfer effectively quenches the fluorescence. Hydrolysis
by recombinant ATX cleaved the phosphodiester bond
resulting in a measurable increase in fluorescence. Because
lysoPLD/ATX is an emerging potential drug target and
biomarker, the substrates provide a simple, sensitive assay
that could be applied to high throughput screening for
diagnosis and drug discovery.
Figure 2. Incubation of FS-2 ([) and FS-3 (9) with recombinant
ATX results in a measurable increase in fluorescence (140 mM
NaCl, 5 mM KCl, 1mM CaCl2, 1 mM MgCl2, 5 mM Tris; pH 8.0,
1 mg/mL fatty acid-free BSA, absorption ) 485 nm, emission )
520 nm, 37 °C).
a more detailed kinetic analysis and comparison with CPF4
and bisNPP will be described in a subsequent paper.17
Although FS-2 and FS-3 are effectively hydrolyzed by
ATX (NPP2), neither would be expected to act as substrates
for NPP1, NPP3, NPP4, or NPP5, which do not hydrolyze
lysophospholipids.18,19 NPP6 has recently been shown to have
lysoPLC activity; however, it is selective for LPC over other
lysophospholipids. Therefore, on the basis of its headgroup
specificity, it may not process FS-2 or FS-3.19 NPP7
Acknowledgment. Work was supported by the National
Cancer Institute (U01CA085133 to Echelon) and the Dutch
Cancer Society (Netherlands Cancer Institute).
Supporting Information Available: Experimental pro-
cedures and characterizations for all new compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(17) van Meeteren, L. A.; Ruurs, P.; Ferguson, C. G.; Prestwich, G. D.;
Ovaa, H.; Moolenaar, W. H. manuscript in preparation.
(18) Gijbers, R.; Aoki, J.; Arai, H.; Bollen, M. FEBS Lett. 2003, 538,
60.
OL060414I
(19) Sakagami, H.; Aoki, J.; Natori, Y.; Nishikawa, K.; Kakehi, Y.;
Natori, Y.; Arai, H. J. Biol. Chem. 2005, 280, 23084.
(20) Duan, R.-D.; Bergman, T.; Xu, N.; Wu, J.; Chen, Y.; Duan, J.;
Nelander, S.; Palmberg, C.; Nilsson, A. J. Biol. Chem. 2003, 278, 38528.
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