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
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