Regioisomeric Family of Novel Fluorescent Substrates for SHIP2

Article abstract of DOI:10.1021/acsmedchemlett.9b00368

SHIP2 (SH2-domain containing inositol 5-phosphatase type 2) is a canonical 5-phosphatase, which, through its catalytic action on PtdInsP₃, regulates the PI3K/Akt pathway and metabolic action of insulin. It is a drug target, but there is limited evidence of inhibition of SHIP2 by small molecules in the literature. With the goal to investigate inhibition, we report a homologous family of synthetic, chromophoric benzene phosphate substrates of SHIP2 that display the headgroup regiochemical hallmarks of the physiological inositide substrates that have proved difficult to crystallize with 5-phosphatases. Using time-dependent density functional theory (TD-DFT), we explore the intrinsic fluorescence of these novel substrates and show how fluorescence can be used to assay enzyme activity. The TD-DFT approach promises to inform rational design of enhanced active site probes for the broadest family of inositide-binding/metabolizing proteins, while maintaining the regiochemical properties of bona fide inositide substrates.

Full text of DOI:10.1021/acsmedchemlett.9b00368

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Letter
A regioisomeric family of novel fluorescent substrates for SHIP2
Gaye White, Christopher Prior, Stephen J. Mills, Kendall Baker, Hayley Whitfield,
Andrew M. Riley, Vasily S. Oganesyan, Barry V. L. Potter, and Charles A. Brearley
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00368 • Publication Date (Web): 18 Oct 2019
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A regioisomeric family of novel fluorescent substrates for SHIP2
Gaye White1†, Christopher Prior2†, Stephen J. Mills3†, Kendall Baker1, Hayley Whitfield1, Andrew M.
Riley3 Vasily S. Oganesyan2*, Barry V.L. Potter3* and Charles A. Brearley1*
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1 School of Biological Sciences, UEA, Norwich Research Park, Norwich NR4 7TJ, UK
2 School of Chemistry, UEA, Norwich Research Park, Norwich NR47TJ, UK
3 Medicinal Chemistry & Drug Discovery, Department of Pharmacology, University of Oxford, Mansfield Road, Oxford
OX1 3QT, UK
† these authors contributed equally to this work
Key Words: Inositol phosphate, 5-phosphatase, SHIP2, TD-DFT, fluorescence, HPLC, ligand
ABSTRACT: SHIP2 (SH2-domain containing inositol 5-phosphatase type 2) is a canonical 5-phosphatase which, through its cata-
lytic action on PtdInsP3, regulates the PI3K/Akt pathway and metabolic action of insulin. It is a drug target but there is limited evi-
dence of inhibition of SHIP2 by small molecules in the literature. With the goal to investigate inhibition, we report a homologous
family of synthetic, chromophoric benzene phosphate substrates of SHIP2 that display the headgroup regiochemical hallmarks of
the physiological inositide substrates that have proved difficult to crystallize with 5-phosphatases. Using time-dependent density
functional theory (TD-DFT), we explore the intrinsic fluorescence of these novel substrates and show how fluorescence can be used
to assay enzyme activity. The TD-DFT approach promises to inform rational design of enhanced active site probes for the broadest
family of inositide-binding / metabolizing proteins, whilst maintaining the regiochemical properties of bona fide inositide sub-
strates.
In eukaryotic cells, many signaling pathways are regulated by
levels of inositol phosphates (inositides), phosphatidylinositol
phosphates (phosphoinositides) and the proteins that are their
cognate binding partners. The balance of these is controlled by
families of kinases and phosphatases that phosphorylate and de-
phosphorylate these molecules at specific positions, designated
locants, of the inositol ring1. SH2-domain containing inositol 5-
phosphatase type 2 (SHIP2) belongs to a family of phosphatases
that hydrolyse the 5-phosphate of inositides and phosphoinosi-
tides. The substrates are known to include inositol pen-
takisphosphates (InsP5), tetrakisphosphates (InsP4), trisphos-
phates (InsP3), phosphatidylinositol 3,4,5-trisphosphate
Previous research for inhibitors of SHIP2 has typically assayed
activity using phosphate release with D-myo-inositol-1,3,4,5-
tetrakisphosphate (Ins(1,3,4,5)P4) or D-myo-phosphatidylinosi-
tol-3,4,5-triphosphate (PtdIns(3,4,5)P3)8. Thus, Suwa et al iden-
tified AS1949490 as a novel, small molecule inhibitor of
SHIP2. In the absence of structural data or evidence of catalytic
modification of the inhibitor, the exact mode of inhibition is not
clear, though from an enzymological perspective the nature of
inhibition is competitive with respect to inositide substrate. It is
possible that within the chemical landscape of inhibition, com-
petitive substrate analog inhibitors will be found. However, fur-
ther mechanistic analysis would require identification of the
products of such reactions, something limited for bona fide sub-
strates by lack of a suitable chromophore in these molecules.
Alternatively, radiolabeled substrates could be used but this
would demand complex synthetic work and HPLC resolution of
substrates and products.
(PtdInsP3)
and
phosphatidylinositol
4,5-bisphosphate
(PtdInsP2) 2.
An understanding of how these enzymes function is important
to pathologies such as diabetes and cancer2. SHIP2 reduces lev-
els of PtdInsP3, while increasing levels of PtdIns(3,4)P2. Con-
sequently, this enzyme regulates the PI3K/Akt pathway which
is linked to cell proliferation and the metabolic action of insu-
lin3,4. SHIP2 is expressed in many cell types and has addition-
ally been linked to regulation of diverse cellular processes such
as calcium signaling, cytoskeletal remodeling, protein traffick-
ing and phagocytosis2, 5. While a mechanistic explanation of in-
hibition of PI3-Kinases has underpinned therapeutic interven-
tions in cancer6,7, therapeutic inhibition of 5-phosphatases lacks
similar foundation.
With a view to finding alternative substrates of 5-phosphatases
that have analytically useful chromophoric properties, we in-
vestigate the benzene polyphosphate compounds shown in Fig-
ure 1. Originally, the benzene phosphate compounds 1 to 6 were
designed as substrate analogs to substitute as ligands to inositol
phosphate binding proteins/enzymes, enabling crystallographic
resolution of protein-ligand complexes and insight into enzyme
inhibition9,10. While simple benzene phosphates appear not to
be substrates of 5-phosphatases9-11 compound 7, 3-OH-
Bz(1,2,4)P3, was synthesized and has been shown by assay of
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Page 2 of 10
release of phosphate to be a substrate for type I myo-inositol
Previously, benzene phosphates have been shown to be inhibi-
1,4,5-triphosphate-5-phosphatase (INPP5A)11. Benzene phos-
phate analogues were also used to uncover mechanistic infor-
mation in co-crystallisation studies with INPP5B12.
tors of INPP5A7, INPP5B12 and SHIP28, with compound 6
Bz(1,2,4,5)P4 yielding IC50 values in single figures and tens of
μM range against Ins(1,4,5)P3 and Ins(1,3,4,5)P4 substrates,
tested at 1 μM and 100 μM concentrations respectively. We also
recently described the use of a fluorescent conjugate of
Ins(1,3,4,5,6)P5 (2-FAM-InsP5, Figure 2) as an active site probe
of inositol pentakisphosphate 2-kinase13 and as an intracellular
probe14. Here, combining the use of two classes of ligand, we
show that displacement of 2-FAM-InsP5 affords assay of ben-
zene phosphate binding to SHIP2 (Figure 2 and Table 1).
1
2
3
4
5
6
7
8
We now show that the chromophoric properties of benzene
phosphates vary depending on the regiochemistry of substitu-
ents around the benzene ring and that several members of this
family have an unanticipated, useful, intrinsic fluorescence.
Alongside conventional spectroscopic measurements, we use
time-dependent density functional theory (TD-DFT) to examine
how the regiochemistry modifies the spectroscopic properties.
Compound 6, Bz(1,2,4,5)P4, proved to be intensely fluorescent,
a property potentially explainable by TD-DFT. It is a tantalizing
observation that the four phosphate groups of this compound
are stereochemically homotopic, remaining indistinguishable
even in the chiral environment of the enzyme active site. Since
5-phosphatases yield only a single product from Ins(1,3,4,5)P4,
should benzene phosphates be substrates we expect a single,
identifiable product 8 5-OH Bz(1,2,4)P3 of dephosphorylation
of compound 6 Bz(1,2,4,5)P4. Compound 8 5-OH Bz(1,2,4)P3
was, however, not synthesized, so in the absence of reference
material we sought to use TD-DFT to predict its fluorescence
with the expectation of being able to use this prediction to iden-
tify whether compound 6 Bz(1,2,4,5)P4 could be a substrate of
SHIP2. We therefore investigated the possibility that benzene
phosphates themselves are substrates for dephosphorylation by
SHIP2 and the chromophoric properties of this class of mole-
cule might allow construction of assays complementary to those
that employ the measurement of released inorganic phosphate.
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All six benzene phosphate derivatives chosen displaced 2nM 2-
FAM-InsP5 from SHIP2 with IC50 values in the low μM range,
the benzene tetrakisphosphates being more effective than ben-
zene trisphosphates, with IC50 values approximately one order
of magnitude smaller than the trisphosphates.
OH
-OOC
O
H
N
O
O
2-O3PO
2-O3PO
2-O3PO
O
2
2-
OPO3
2-
OPO3
2-FAM-InsP5
300
200
100
Bz(1,2,3)P3
Bz(1,3,5)P3
Bz(1,2,4)P3
Bz(1,2,3,4)P4
Bz(1,2,4,5)P4
Bz(1,2,3,5)P4
0.1
1
10
100
Displacing ligand (mM)
Figure 2 Displacement of 2-FAM-InsP5 from SHIP2 by ben-
zene phosphates. The experiments used 2nM 2-FAM-InsP5 and
1μM SHIP2. Results shown as mean of triplicate experiments
(results with standard deviations given in Table 1).
Figure 1. Structures of benzene phosphates used in this study
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ACS Medicinal Chemistry Letters
Table 1. Comparison of published IC50 values for inhibition of 5-phosphatases by benzene phosphates to experimentally determined
IC50 values found for displacement of 2-FAM-InsP5 from SHIP2.
1
2
3
4
5
Compound
IC50 (μM)
IC50 (μM)
Ins(1,3,4,5)P4
IC50 (μM)
IC50 (μM)
2-FAM-
Ins(1,4,5)P3 Ins(1,4,5)P3
InsP5 SHIP2 SHIP2 10, 12
INPP5A 9
INPP5B 12
this study
6
7
8
9
Bz(1,2,3)P3
Bz(1,3,5)P3
Bz(1,2,4)P3
14.23 ± 0.1
11.25 ± 2.9
7.02 ± 0.05
>1000
86 ± 28
16 ± 9
14 ± 9
98 ± 16
78 ± 50
4 ± 2
33.5 ± 6.8
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Bz(1,2,3,4)P4 2.78 ± 0.59
Bz(1,2,3,5)P4 1.01 ± 0.43
Bz(1,2,4,5)P4 1.02 ± 0.1
69.3±15.410
108.3± 20.312
6.3 ± 0.8
Most commonly, 5-phosphatase activity is assayed as the re-
lease of inorganic phosphate from Ins(1,3,4,5)P4 or
PtdIns(3,4,5)P3. We assayed the ability of SHIP2 to release
phosphate from inositol phosphates and also benzene phos-
phates, and test whether benzene phosphates are substrates. We
tested whether by virtue of the lower pKa of the hydroxyl of a
theoretical dephosphorylated benzene phosphate (leaving
group)15,16, they might be better substrates than simple inositol
phosphate substrate (Figure 3).
Ins(1,3,4,5)P4 and InsP6 were included as positive and negative
controls. Phosphate release was determined with molybdenum
blue, using ferrous sulphate as a reducing agent17. While slower
than the reaction with Ins(1,3,4,5)P4, phosphatase activity was
noted with all compounds except InsP6, a substrate for
phytases18 but not for inositol polyphosphate 5-phosphatases.
As the benzene phosphates are poorer substrates than
Ins(1,3,4,5)P4, the rate of de-phosphorylation must be influ-
enced by factors other than just the pKa of the leaving group. In
an earlier study11, we postulated that OH groups in the substrate
are likely to have a mechanistic role, showing that during de-
phosphorylation of compound 7 3-OH-Bz(1,2,4)P3 by INPP5A,
stabilization of the phenolic OH proton accompanied de-proto-
nation of the phosphate groups. In order to test this theory, we
0.15
0.10
0.05
0.00
compared phosphate release from compound
7 3-OH-
Bz(1,2,4)P3 and compound 4 Bz(1,2,3,4)P4 (Figure S1). The
rate determined for compound 7 3-OH-Bz(1,2,4)P3 was almost
twice that observed for compound 4 Bz(1,2,3,4)P4. However,
this rate is comparable to that observed for compound 5
Bz(1,2,3,5)P4, suggesting that the regiochemistry of the ring
substitution may also have an influence.
Having gained evidence for binding of benzene phosphates to
SHIP2 and their catalytic processing by SHIP2, we speculated
that the spectroscopic properties of benzene phosphates might
be exploited to follow catalysis. Excitation and emission scans
of acetonitrile solutions of compound 4 Bz(1,2,3,4)P4, com-
pound 5 Bz(1,2,3,5)P4, compound 6 Bz(1,2,4,5)P4 and trypto-
phan were measured and the spectral details compared to those
predicted by TD-DFT (Figure 4 and Tables S1 and S2). Spectral
predictions for compound 8 5-OH-Bz(1,2,4)P3 are included in
the table, but the pure compound was not available to test. The
theoretical methods employ approaches used in TD-DFT stud-
ies of tryptophan19,20. A detailed discussion of the TD-DFT
methods and results is included in Supporting Information.
P 4
P 6
P 3
P 3
P 3
P 4
P 4
P 4
)
)
)
)
)
)
s
s
3
5
4
(
4
(
5
(
5
,
n
n
I
,
,
,
,
,
I
2
3
2
3
3
4
,
,
,
,
,
,
1
1
1
(
2
2
2
,
,
,
(
(
z
z
z
1
1
1
B
B
B
z
z
z
B
B
B
Figure 3. Benzene phosphates as substrates of SHIP2. Com-
pounds tested at 100μM. For Ins(1,3,4,5)P4 and InsP6, the con-
centration of SHIP2 was 1μM and for the benzene phosphates,
4μM, with triplicate reactions run for 10min at 30°C.
Significantly, on excitation at 280nm, the measured fluores-
cence of compound 6 Bz(1,2,4,5)P4, was 30 times more intense
than that of the next most fluorescent benzene tetrakisphos-
phate, compound 5 Bz(1,2,3,5)P4, and approximately 10% of
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ACS Medicinal Chemistry Letters
that of tryptophan (Figure 4). These observations match the
Page 4 of 10
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5
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8
trends predicted by TD-DFT (Tables S1 and S2). The calcula-
tions for compound 8 5-OH-Bz(1,2,4)P3 predict that the excita-
tion maximum of 5-OH-Bz(1,2,4)P3 is 6nm greater than that of
compound 6 Bz(1,2,4,5)P4, a prediction confirmed by empirical
measurement (Figure 5 and Table S1). Similarly, TD-DFT pre-
dicts the emission maximum for compound 8 5-OH-Bz(1,2,4)P3
is 25nm greater than that of compound 6 Bz(1,2,4,5)P4 (Figure
5 and Table S2).
A
0 hours
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10 hours
A
B
12 hours
0
10
20
30
40
Retention Time (min)
300 320 340 360 380 400
Emission (nm)
220 240 260 280 300
Excitation (nm)
B
C
Figure 4. Fluorescence spectra of 500μM Bz(1,2,3,4)P4 (cyan
line), 500μM Bz(1,2,3,5)P4 (green line), 100μM Bz(1,2,4,5)P4
(blue line), 10μM tryptophan (black dotted line) in acetonitrile.
(A) excitation scans determined at emission maxima, (B) emis-
sion scans excited at 280nm.
As TD-DFT predicts that compound 6 Bz(1,2,4,5)P4 and its hy-
drolysis product compound 8 5-OH-Bz(1,2,4)P3 show signifi-
cant fluorescence intensity, we devised an HPLC separation
that could be used with fluorescence detection for benzene
phosphates. First, we established separations following UV ab-
sorbance (Figure S2). HPLC resolved all the discrete benzene
trisphosphates and tetrakisphosphates used in this study.
Compound 6 Bz(1,2,4,5)P4 that eluted at approximately 37
minutes could also be detected by fluorescence with excitation
at 280nm and emission at 330nm. In the first instance we
wanted to use the HPLC method to investigate whether SHIP2
could convert compound 6 Bz(1,2,4,5)P4 into compound 8 5-
OH-Bz(1,2,4)P3. Therefore, we selected conditions that would
limit the extent of reaction to a predominant single dephosphor-
ylation. Extended incubation of 100μM of compound 6
Bz(1,2,4,5)P4 with 100nM SHIP2 allowed us to monitor the
progression of the reaction without interference from other flu-
orescent products as judged by HPLC (Figure 5A). Over a pe-
riod of 12h, the fluorescence intensity of the parent peak at
37min diminished correlating with the appearance of an earlier-
eluting peak at 30min (Figure 5A). As we did not have the as-
sumed product, compound 8 5-OH-Bz(1,2,4)P3, to confirm the
identity of the peak at 30 minutes (Figure 5A), we sought con-
firmation by recording fluorescence excitation and emission
spectra of the accumulated products (Figures 5B and 5C).
250 260 270 280 290 300
300 320 340 360 380 400
Excitation (nm)
Emission (nm)
SHIP2
Red Shift on dephosphorylation
Excitation
6 nm
7 nm
Emission
25 nm
25 nm
Predicted
Observed
Figure 5. (A) HPLC analysis of reaction of Bz(1,2,4,5)P4
(100μM) incubated with SHIP2 (100nM) at 16°C for 0h (blue
line), 10h (amber line) and 12h (red line). Substrate and prod-
ucts were detected by fluorescence (excitation at 280 nm, emis-
sion at 330 nm). (B) & (C) Fluorescence of the same samples
diluted 100-fold in acetonitrile. 100nM SHIP2 alone shown as
dark green line on baseline. (B) excitation scans with emission
at 330nm, (C) emission scans excited at 280nm.
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Samples analyzed in Figure 5A were diluted 100-fold into ace-
tonitrile and fluorescence spectra were recorded (Figures 5B
1
2
3
4
5
6
7
8
Time
and 5C). As the proportion of the product increased, the red
shifts in the fluorescence spectra increased correspondingly.
After 12h incubation, the excitation maximum of the assumed
product, compound 8 5-OH-Bz(1,2,4)P3, was 7nm greater than
that of the starting material (Figure 5B). This compares favora-
bly to the shift of +6 nm predicted by TD-DFT for compound 6
Bz(1,2,4,5)P4 converting to compound 8 5-OH-Bz(1,2,4)P3
(Table S1). The emission maximum of the product was 25 nm
greater than that of the starting material (Figure 5C). This also
compares favorably to the predicted shift of +25 nm for com-
pound 6 Bz(1,2,4,5)P4 converting to compound 8 5-OH-
Bz(1,2,4)P3 (Table S2). As discussed in Supporting Infor-
mation, the predicted transition energies for excitation and
emission differ systematically from the experimentally deter-
mined values so that variations between compounds can be de-
fined in terms of wavelength shifts. A similar observation has
A
1500
1000
500
0
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Wavelength (nm)
B
1600
1400
previously been noted for this type of TD-DFT analysis19,20
.
+ Ins(1,3,4,5)P4
From the close match between the predicted and observed
wavelength shifts we can conclude that compound 8 5-OH-
Bz(1,2,4)P3 is formed when compound 6 Bz(1,2,4,5)P4 is incu-
bated with SHIP2 under these conditions.
+ AS1949490
No additives
In order to investigate further the effects of SHIP2 on com-
pound 6 Bz(1,2,4,5)P4, 100M of the substrate was incubated
for 2h with increasing amounts of SHIP2. This demonstrated
that the conversion of compound 6 Bz(1,2,4,5)P4 into its first
hydrolysis product, compound 8 5-OH-Bz(1,2,4)P3, is depend-
ent on the SHIP2 concentration and compound 6 Bz(1,2,4,5)P4
alone is stable under these conditions (Figure S3). Longer incu-
bation and use of tandem UV-fluorescence detection resulted in
a more complicated product profile with peaks of increased ab-
sorbance: fluorescence ratio at 30, 25, 23 and 13min (Figure
S4). This is consistent with successive de-phosphorylation gen-
erating a sequence of different hydroxybenzene phosphate com-
pounds with different elution profiles as observed for inositol
phosphates21.
0
20
40
60
80
100
120
Time (mins)
Figure 6. (A) Change in fluorescence of compound 6
Bz(1,2,4,5)P4 (100M) incubated with SHIP2 (1M) at 23C
recording emission scans excited at 280nm over a period of 2
hours. (B) Change in emission intensity at 325nm with time for
compound 6 Bz(1,2,4,5)P4 incubated with SHIP2; with no other
additives (dark blue line), with Ins(1,3,4,5)P4 (50M) (peach
line), with SHIP2 inhibitor AS1949490 (100M) (light blue
line).
While there is much to take account of when designing such an
experiment, we have proved the utility of TD-DFT to select
from a regioisomeric family of benzene tris- and tetrakisphos-
phates for the compound with the best fluorescent properties to
monitor 5-phosphatase, specifically SHIP2, activity. Interest-
ingly, benzene phosphates appear to undergo successive
dephosphorylations with SHIP2, in contrast to the single 5-
dephosphorylation of inositol phosphate substrates. By judi-
cious choice of a symmetrical tetrakisphosphate and careful ti-
tration of enzyme, we were able to limit the extent of reaction
to a predominant single dephosphorylation. Interestingly, to-
wards the end of the experiment shown in Figure 6A there is a
slight shift to shorter wavelength again, most likely reflecting
further de-phosphorylation of the 5-OH-Bz(1,2,4)P3 product.
The change in fluorescence that accompanies the conversion of
substrate to product can be used to develop a real-time fluores-
cence-based assay to follow de-phosphorylation of compound
6 Bz(1,2,4,5)P4 by the exemplar 5-phosphatase SHIP2 (Figure
6). In the absence of competing additives, the fluorescence in-
tensity at 325nm reduces and the maximum shifts to a longer
wavelength as the reaction between SHIP2 and compound 6
Bz(1,2,4,5)P4 progresses (Figure 6A). Figure 6B traces how the
emission intensity at 325 nm (the maximum for compound 6
Bz(1,2,4,5)P4) decreases with time. It also shows how this de-
crease is affected by additives that are reported to be substrates
or inhibitors of SHIP2 activity. The rate of the intensity de-
crease slows down considerably in the presence of the natural
substrate Ins(1,3,4,5)P4 which, as shown in Figure 3, is 10 times
more active in terms of phosphate release than any of the ben-
zene phosphates. In the presence of the SHIP2 inhibitor
AS1949490, there is an initial decrease in fluorescence intensity
that matches that of the reaction with no additives indicating
that, in the initial stages under these conditions, we are not see-
To explore further the catalytic flexibility engendered in phos-
phate-substituted benzenes, we also sought to define the prod-
ucts of successive dephosphorylation of the more weakly fluo-
rescent compound 4, Bz(1,2,3,4)P4 (Figure S5), for which a
number of potential dephosphorylation products are available.
Accordingly, we included compound 7 3-OH-Bz(1,2,4)P3, and
the newly synthesized compounds 9 1,2-Di-OH-Bz(3,4)P2 and
10 1,3-Di-OH-Bz(2,4)P2 in our analysis (Full details and struc-
tures of these compounds are given in Supporting Infor-
mation)]. We did not detect the accumulation of compound 7,
ing inhibition of SHIP2 activity with compound
6
Bz(1,2,4,5)P4. This experiment is not an exhaustive analysis of
the substrates competing for SHIP2 activity but serves to
demonstrate the utility of benzene phosphate fluorescence in
this context.
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3-OH-Bz(1,2,4)P3 from compound 4 Bz(1,2,3,4)P4. This does SopB. The demonstration that they are substrates and can be
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not rule out its production or that of 1-OH-Bz(2,3,4)P3 as there
is the possibility that the first product of hydrolysis is rapidly
dephosphorylated to Di-OH-BzP2 products. Indeed, compound
9 1,2-Di-OH-Bz(3,4)P2 = 3,4-Di-OH-Bz(1,2)P2 was generated
by SHIP2 from compound 4 Bz(1,2,3,4)P4 (Figure S4) and,
from Figure S1, we can see that compound 7 3-OH-Bz(1,2,4)P3
is a better phosphate-releasing substrate than compound 4
Bz(1,2,3,4)P4. Consistently, SHIP2 action on compound 7 3-
OH-Bz(1,2,4)P3 yielded multiple products including compound
9 1,2-Di-OH-Bz(3,4)P2 = 3,4-Di-OH-Bz(1,2)P2, but not com-
pound 10 1,3-Di-OH-Bz(2,4)P2 = 2,4-Di-OH-Bz(1,3)P2 (Fig-
ure S4). [2,3-Di-OH-Bz(1,4)P2 was not available to test]. The
intricacies of this analysis demonstrate further the advantages
of a planned approach to selecting substrates with the most suit-
able properties for probing selected mechanisms of protein ac-
tivity.
used for real-time assays affords great opportunity for study of
allosteric regulation of catalytic activity by ligand binding to
distal domains of the full-length protein.
Because of its relatively strong absorption and fluorescence
emission, compound 6 Bz(1,2,4,5)P4 is shown to be a promising
spectroscopic probe for inositol 5-phosphatase(s). It may also
be of use for proteins lacking catalytic activity since, as demon-
strated, it is good ligand of inositol phosphate-binding sites. Be-
yond this, the combination of TD-DFT and enzymological ap-
proaches could direct the synthesis of new probes for study of
specific aspects of protein structure and function. For real time
assays, designed to screen potential inhibitors of protein activ-
ity, there is opportunity to design chromophores with enhanced,
perhaps further red-shifted, fluorescence. Spectral separation
from tryptophan, either in excitation and/or emission, might en-
able polarization-based approaches to inhibitor screening of in-
ositol phosphate-metabolizing enzymes as we have described
with 2-FAM-InsP5 for IPK113, but without the potential con-
straints of bulky fluorophore substituents. Probes might be syn-
thesized to retain the ligand co-ordination of bona fide sub-
strates and/or might afford a range of binding constants tailored
for particular experimental scenarios, cf. ion-sensing fluores-
cent probes. Thus, we envisage our methods will enable predic-
tion and testing of poly-substituted benzenes that retain the re-
giochemistry of a favored ligand whilst building in the spectro-
scopic opportunity of substitution with other functionalities.
The diversification of simple aryl ligands to include other func-
tionalities may further find use as structure-stabilizing ligands
enabling crystallization of recalcitrant inositol-related protein
targets12. In summary, there is wide-ranging potential in this
field for the application of TD-DFT to aid decisions over probe
synthesis and experiment design.
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To summarize, with interest in the utility of benzene phosphates
as ligands of diverse inositide / phosphoinositide-binding pro-
teins / enzymes, we have shown that, uniquely among charac-
terized ligands of 5-phosphatases, benzene phosphates have the
properties of bona fide substrate analogs. Indeed, benzene tris-
and tetrakisphosphates are inhibitors of Type I and Type II ino-
sitol polyphosphate 5-phosphatases9,12, while compound 4
Bz(1,2,3,4)P4 is a tight binding ligand of the PH domain of
PKB/Akt22. Compound 6 Bz(1,2,4,5)P4 is an inhibitor of
SHIP210 and a structure was solved for a binary complex with
INPP5B12. Here we show that benzene phosphates are surro-
gate substrates of the canonical 5-phosphatase SHIP2 by virtue
of their regiochemistry which mimics well the stereoisomerism,
but not enantiomers, of inositides and phosphoinositides.
Clearly these compounds could find utility with a range of ino-
sitol (phosphate) phosphatases beyond 5-phosphatases, e.g., 3-
phosphatases typified by PTEN or 4-phosphatases such as
ASSOCIATED CONTENT
Supporting Information:
Experimental Procedures; including chemical syntheses, protein purification, analytical methods and TD-DFT calculations.
Figure S1: Assay of benzene phosphates and hydroxybenzene phosphates as substrates of SHIP2
Figure S2: HPLC separation of benzene phosphates
Figure S3: HPLC of SHIP2 action on compound 6 Bz(1,2,4,5)P4
Figure S4: Tandem UV-fluorescence HPLC assay of SHIP2 action on compound 6 Bz(1,2,4,5)P4
Figure S5: HPLC separation of compound 4 Bz(1,2,3,4)P4, compound 7 3-OH-Bz(1,2,3)P3, compound 9 1,2-Di-OH-Bz(3,4)P2 and
compound 10 1,3-Di-OH-Bz(2,4)P2
TD_DFT: Results, Tables and Figures
AUTHOR INFORMATION
Corresponding Authors
*Tel; +44 1603 592197. Email: c.brearley@uea.ac.uk
Tel; +44 1865 271945. Email: barry.potter@pharm.ox.ac.uk
Tel; +44 1603 592698. Email: v.oganesyan@uea.ac.uk
ORCID
Charles A Brearley: 0000-0001-6179-9109
Barry V L Potter: 0000-0001-3255-9315
Vasily S Oganesyan: 0000-0002-8738-1146
Author Contributions
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G.W., K.B., H.W., C.P., S.J.M. and C.A.B. performed experiments.
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G.W., V.O., S.J.M., A.M.R., B.V.L.P., and C.A.B. designed the study and wrote the manuscript.
Funding
Funding supporting this study was obtained by C.A.B. (BBSRC BB/N002024/1 with contribution from AB Vista), V.S.O. (EPSRC
EP/P007554/1) and B.V.L.P. (Wellcome Trust Senior Investigator Grant 101010). K.B acknowledges support from AB Vista.
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ABBREVIATIONS
2-FAM-InsP5, 2-O-(2-(5-fluoresceinylcarboxy)-aminoethyl)-myo-inositol 1,3,4,5,6-pentakisphosphate (triethylammonium salt)
3-OH-Bz(1,2,4)P3, 3-hydroxybenzene 1,2,4-trisphosphate
5-OH-Bz(1,2,4)P3, 5-hydroxybenzene 1,2,4-trisphosphate
1,2-Di-OH-Bz(3,4)P2, 1,2-Dihydroxybenzene-3,4-bisphosphate
1,3-Di-OH-Bz(2,4)P2, 1,3-Dihydroxybenzene-2,4-bisphosphate
2,3-Di-OH-Bz(1,4)P2, 2,3-Dihydroxybenzene-1,4-bisphosphate
BzP, benzene phosphate
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Bz(1,2,3)P3, benzene 1,2,3-trisphosphate
Bz(1,2,4)P3, benzene 1,2,4-trisphosphate
Bz(1,3,5)P3, benzene 1,3,5-trisphosphate
Bz(1,2,3,4)P4, benzene 1,2,3,4-tetrakisphosphate
Bz(1,2,3,5)P4, benzene 1,2,3,5-tetrakisphosphate
Bz(1,2,4,5)P4, benzene 1,2,4,5-tetrakisphosphate
EDTA, ethylenediamine tetra-acetic acid
HEPES, 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid
His, histidine
HOMO, highest occupied molecular orbital
HPLC, high-pressure liquid chromatography
IC50, half-maximal inhibitory concentration
INPP5A, type I inositol 5-phosphatase
INPP5B, type II inositol 5-phosphatase
INPP5E, inositol polyphosphate 5-phosphatase E
Ins(1,4,5)P3, inositol 1,4,5-trisphosphate
Ins(1,3,4,5)P4, inositol 1,3,4,5-tetrakisphosphate
LUMO, lowest unoccupied molecular orbital
OCRL-1, Lowe oculocerebrorenal syndrome protein (INPP5F)
PKB, Protein Kinase B
PtdIns(3,4)P2, phosphatidylinositol 3,4-bisphosphate
PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate
PtdIns(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate
SYNJ1, synaptojanin-1
SYNJ2, synaptojanin-2
SHIP1, SH2-domain containing inositol 5-phosphatase type 1
SHIP2, SH2-domain containing inositol 5-phosphatase type 2
TCEP, tris(2-carboxyethyl) phosphine
TD-DFT, time-domain density functional theory
TEV, Tobacco Etch Virus
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A regioisomeric family of novel fluorescent substrates for SHIP2
Gaye White1†, Christopher Prior2†, Stephen J. Mills3†, Kendall Baker1, Hayley Whitfield1, Andrew M.
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Riley3 Vasily S. Oganesyan2*, Barry V.L. Potter3* and Charles A. Brearley1*
For Table of Contents Only
1.
Abel, K.; Anderson, R. A.; Shears, S. B., Phosphatidylinositol and inositol phosphate metabolism.
J. Cell. Sci. 2001, 114 (Pt 12), 2207-8.
2.
Ooms, L. M.; Horan, K. A.; Rahman, P.; Seaton, G.; Gurung, R.; Kethesparan, D. S.; Mitchell, C.
A., The role of the inositol polyphosphate 5-phosphatases in cellular function and human disease.
Biochem. J. 2009, 419 (1), 29-49.
3.
Zhang, J.; Liu, Z.; Rasschaert, J.; Blero, D.; Deneubourg, L.; Schurmans, S.; Erneux, C.; Pesesse,
X., SHIP2 controls PtdIns(3,4,5)P(3) levels and PKB activity in response to oxidative stress. Cellular
Signalling 2007, 19 (10), 2194-200.
4.
Le Coq, J.; Camacho-Artacho, M.; Velazquez, J. V.; Santiveri, C. M.; Gallego, L. H.; Campos-
Olivas, R.; Dolker, N.; Lietha, D., Structural basis for interdomain communication in SHIP2 providing
high phosphatase activity. eLife 2017, 6.
5.
Thomas, M. P.; Erneux, C.; Potter, B. V. L., SHIP2: Structure, Function and Inhibition.
ChemBioChem 2017, 18 (3), 233-247.
6.
Dalton, S. E.; Dittus, L.; Thomas, D. A.; Convery, M. A.; Nunes, J.; Bush, J. T.; Evans, J. P.;
Werner, T.; Bantscheff, M.; Murphy, J. A.; Campos, S., Selectively Targeting the Kinome-Conserved
Lysine of PI3Kdelta as a General Approach to Covalent Kinase Inhibition. J. Am. Chem. Soc. 2018, 140
(3), 932-939.
8
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7.
Miller, M. S.; Thompson, P. E.; Gabelli, S. B., Structural Determinants of Isoform Selectivity in
1
2
PI3K Inhibitors. Biomolecules 2019, 9 (3), 82.
3
4
8.
Suwa, A.; Yamamoto, T.; Sawada, A.; Minoura, K.; Hosogai, N.; Tahara, A.; Kurama, T.;
Shimokawa, T.; Aramori, I., Discovery and functional characterization of a novel small molecule inhibitor
of the intracellular phosphatase, SHIP2. Brit. J. Pharmacol. 2009, 158 (3), 879-887.
5
6
7
8
9
9.
Mills, S. J.; Vandeput, F.; Trusselle, M. N.; Safrany, S. T.; Erneux, C.; Potter, B. V. L., Benzene
polyphosphates as tools for cell signalling: inhibition of inositol 1,4,5-trisphosphate 5-phosphatase and
10
11
12
13
14
15
16
17
18
19
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47
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50
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53
54
55
56
57
58
59
60
interaction with the PH domain of protein kinase Balpha. ChemBioChem 2008, 9 (11), 1757-66.
10.
Mills, S. J.; Persson, C.; Cozier, G.; Thomas, M. P.; Tresaugues, L.; Erneux, C.; Riley, A. M.;
Nordlund, P.; Potter, B. V. L., A synthetic polyphosphoinositide headgroup surrogate in complex with
SHIP2 provides a rationale for drug discovery. ACS Chem. Biol. 2012, 7 (5), 822-8.
11.
Mills, S. J.; Dozol, H.; Vandeput, F.; Backers, K.; Woodman, T.; Erneux, C.; Spiess, B.; Potter,
B. V. L., 3-hydroxybenzene 1,2,4-trisphosphate, a novel second messenger mimic and unusual substrate
for type-I myo-inositol 1,4,5-trisphosphate 5-phosphatase: Synthesis and Physicochemistry.
ChemBioChem 2006, 7 (11), 1696-706.
12.
Mills, S. J.; Silvander, C.; Cozier, G.; Tresaugues, L.; Nordlund, P.; Potter, B. V. L., Crystal
Structures of Type-II Inositol Polyphosphate 5-Phosphatase INPP5B with Synthetic Inositol
Polyphosphate Surrogates Reveal New Mechanistic Insights for the Inositol 5-Phosphatase Family.
Biochemistry 2016, 55 (9), 1384-1397.
13.
Whitfield, H.; Gilmartin, M.; Baker, K.; Riley, A. M.; Godage, H. Y.; Potter, B. V. L.; Hemmings,
A. M.; Brearley, C. A., A Fluorescent Probe Identifies Active Site Ligands of Inositol Pentakisphosphate
2-Kinase. J. Med. Chem. 2018, 61 (19), 8838-8846.
14.
Riley, A. M.; Windhorst, S.; Lin, H. Y.; Potter, B. V. L., Cellular internalisation of an inositol
phosphate visualised by using fluorescent InsP5. ChemBioChem 2014, 15 (1), 57-67.
15.
16.
Kirby, A. J.; Nome, F., Fundamentals of phosphate transfer. Acc Chem Res 2015, 48 (7), 1806-14.
Pabis, A.; Duarte, F.; Kamerlin, S. C., Promiscuity in the Enzymatic Catalysis of Phosphate and
Sulfate Transfer. Biochemistry 2016, 55 (22), 3061-81.
17.
Taussky, H. H.; Shorr, E., A Microcolorimetric Method for the Determination of Inorganic
Phosphorus. J. Biol. Chem. 1953, 202 (2), 675-685.
18.
Gruninger, R. J.; Dobing, S.; Smith, A. D.; Bruder, L. M.; Selinger, L. B.; Wieden, H. J.;
Mosimann, S. C., Substrate binding in protein-tyrosine phosphatase-like inositol polyphosphatases. J.
Biol. Chem. 2012, 287 (13), 9722-30.
19.
Caricato, M.; Mennucci, B.; Tomasi, J.; Ingrosso, F.; Cammi, R.; Corni, S.; Scalmani, G.,
Formation and relaxation of excited states in solution: a new time dependent polarizable continuum model
based on time dependent density functional theory. J. Chem. Phys. 2006, 124 (12), 124520-1-13.
20.
Al-Soliemy, A. M.; Osman, O. I.; Hussein, M. A.; Asiri, A. M.; El-Daly, S. A., Fluorescence,
Photophysical Behaviour and DFT Investigation of E,E-2,5-bis[2-(3-pyridyl)ethenyl]pyrazine (BPEP). J.
Fluoresc. 2016, 26 (4), 1199-209.
9
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2
3
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21.
Kuo, H. F.; Hsu, Y. Y.; Lin, W. C.; Chen, K. Y.; Munnik, T.; Brearley, C. A.; Chiou, T. J.,
Arabidopsis inositol phosphate kinases, IPK1 and ITPK1, constitute a metabolic pathway in maintaining
phosphate homeostasis. Plant J. 2018, 95, 613, DOI: 10.1111/tpj.13974
5
6
7
8
9
22.
Mills, S. J.; Komander, D.; Trusselle, M. N.; Safrany, S. T.; van Aalten, D. M.; Potter, B. V. L.,
Novel inositol phospholipid headgroup surrogate crystallized in the pleckstrin homology domain of
protein kinase Balpha. ACS Chemical Biology 2007, 2 (4), 242-6.
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