5-Stabilized phosphatidylinositol 3,4,5-trisphosphate analogues bind Grp1 PH, inhibit phosphoinositide phosphatases, and block neutrophil migration

Article abstract of DOI:10.1002/cbic.200900545

Metabolically stabilized analogues of PtdIns(3,4,5)P₃ have shown long-lived agonist activity for cellular events and selective inhibition of lipid phosphatase activity. We describe an efficient asymmetric synthesis of two 5-phosphatase-resistan

Full text of DOI:10.1002/cbic.200900545

DOI: 10.1002/cbic.200900545
5-Stabilized Phosphatidylinositol 3,4,5-Trisphosphate
Analogues Bind Grp1 PH, Inhibit Phosphoinositide
Phosphatases, and Block Neutrophil Migration
Honglu Zhang,[a] Ju He,[b] Tatiana G. Kutateladze,[b] Takahiro Sakai,[c] Takehiko Sasaki,[c]
Nicolas Markadieu,[d] Christophe Erneux,[d] and Glenn D. Prestwich*[a]
Metabolically stabilized analogues of PtdIns(3,4,5)P3 have
shown long-lived agonist activity for cellular events and selec-
tive inhibition of lipid phosphatase activity. We describe an ef-
ficient asymmetric synthesis of two 5-phosphatase-resistant an-
alogues of PtdIns(3,4,5)P3, the 5-methylene phosphonate (MP)
and 5-phosphorothioate (PT). Furthermore, we illustrate the
biochemical and biological activities of five stabilized PtdIns-
(3,4,5)P3 analogues in four contexts. First, the relative binding
affinities of the 3-MP, 3-PT, 5-MP, 5-PT, and 3,4,5-PT3 analogues
to the Grp1 PH domain are shown, as determined by NMR
spectroscopy. Second, the enzymology of the five analogues is
explored, showing the relative efficiency of inhibition of SHIP1,
SHIP2, and phosphatase and tensin homologue deleted on
chromosome 10 (PTEN), as well as the greatly reduced ability
of these phosphatases to process these analogues as sub-
strates as compared to PtdIns(3,4,5)P3. Third, exogenously de-
livered analogues severely impair complement factor C5a-
mediated polarization and migration of murine neutrophils. Fi-
nally, the new analogues show long-lived agonist activity in
mimicking insulin action in sodium transport in A6 cells.
Introduction
The phosphoinositide 3-kinase (PI 3-K) signaling pathway con-
tains important therapeutic targets in human pathophysiolo-
gy.[1,2] Phosphatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P3) is
a ubiquitous signaling lipid found in higher eukaryotic cells[3]
and activates a plethora of downstream cellular processes.[4]
These signaling events include cell proliferation and transfor-
mation,[5] cell shape and motility,[6] and insulin action and alter-
ation of glucose transport.[7] PtdIns(3,4,5)P3-regulated signaling
can be modulated by the lipid 3-phosphatase PTEN[8] and SH2
domain-containing inositol 5-phosphatases SHIP1 and SHIP2.[9]
Indeed, PTEN and SHIP1/2 are crucial regulators of PI 3-kinase
signaling in T-lymphocytes,[10] drug-discovery targets in type 2
diabetes[11] and intimately involved in signaling networks for a
myriad of other disease states.[12]
tant phosphomimetics that show greatly reduced rates of
enzyme-mediated hydrolysis.[15] However, the replacement of
P=O by P=S also affects the pKa of the phosphate and removes
a
H-bond acceptor.[16,17] Indeed, we demonstrated that
PtdIns(3)PT had reduced binding activity for cognate
PtdIns(3)P-selective FYVE and PX domain binding proteins; this
could be attributed to reduced H-bonding.[18] We also ob-
served that a PT analogue of PtdIns(5)P was a long-lived ago-
nist for chromatin remodeling,[19] and a tris(PT) cyclopentyl an-
alogue[20] of Ins(1,4,5)P3 was a long-lived agonist activities for
[a] Dr. H. Zhang, Prof. Dr. G. D. Prestwich
Department of Medicinal Chemistry, The University of Utah
419 Wakara Way, Suite 205, Salt Lake City, UT 84108-1257 (USA)
Fax: (+1)801-585-9053
A metabolically stabilized (ms) analogue of PtdIns(3,4,5)P3
that resists lipid 3- and 5-phosphatases would have numerous
applications in understanding the role of PtdIns(3,4,5)P3 in cell
physiology. The ms-PtdIns(3,4,5)P3 analogues could separate
the activation of signal transduction from the degradation of
the signal by phosphatase action in cells. This chemical biology
approach to the dissection of the PI 3-K pathway would be
complementary to the use of siRNA knockdowns or genetic
knockouts for PTEN and SHIP.
E-mail: gprestwich@pharm.utah.edu
[b] Dr. J. He, Prof. Dr. T. G. Kutateladze
Department of Pharmacology,
University of Colorado Denver, School of Medicine
12801 East 17th Ave, Aurora, CO 80045-0511 (USA)
[c] Dr. T. Sakai, Prof. Dr. T. Sasaki
Division of Microbiology, Department of Pathology and Immunology,
Akita University School of Medicine
1-1-1 Hondo, 010-8543, Akita (Japan)
[d] Dr. N. Markadieu, Prof. Dr. C. Erneux
Department of Cell Physiology and
We recently described the preparation and activity of the 3-
phosphatase-resistant, metabolically stabilized analogues 1
and 2 (Scheme 1) of PtdIns(3,4,5)P3, which were both stable to
degradation by PTEN and acted as inhibitors of PTEN.[13] One
of these analogues incorporated a single phosphorothioate
(PT) substituent, and an additional study examined the chemis-
try and biological activity of the 3,4,5-trisphosphorothioate an-
alogue (3) of PtdIns(3,4,5)P3.[14] Phosphorothioates are impor-
Institut de Recherche Interdisciplinaire (IRIBHM)
Universitꢀ Libre de Bruxelles, Campus Erasme
Route de Lennik 808, 1070 Bruxelles, (Belgium)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.200900433: complete experimental details for
synthesis and characterization of new compounds with chemical structures,
inhibition of PTEN by metabolically stabilized analogues, Akt activation by
analogues.
388
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2010, 11, 388 – 395

Phosphatidylinositol Trisphosphate Analogues
Scheme 1. Analogues of phosphatidylinositol 3,4,5-trisphosphate synthesized from d-myo-inositol.
the IP3 receptor. We hypothesized that 5-PT and 5-MP ana-
logues of PtdIns(3,4,5)P3 could be either antagonists or long-
lived PtdIns(3,4,5)P3 agonists, as they would be more slowly
dephosphorylated by the 5-phosphatase SHIP and could po-
tentially block the normal receptor-mediated signaling involv-
ing PtdIns(3,4,5)P3. We also wished to test whether the 5-PT
and 5-MP modifications could alter the processing of PtdIns-
(3,4,5)P3 by the 3-phosphatase PTEN.
been synthesized previously. The synthesis of 5-ms-PtdIns-
(3,4,5)P3 required selective protection of the 5-OH and the use
of enantiomerically pure myo-inositol derivatives. A number of
strategies have been developed for the synthesis of optically
pure phosphoinositides, either using the enantiopure natural
precursors, such as d-glucose[21] or quinic acid.[22] Kinetic reso-
lution or desymmetrization by enantioselective enzymatic acy-
lation, nonenzymatic phosphorylation of myo-inositol deriva-
tives,[23-25] and the separation of diastereomeric derivatives of
myo-inositol with chiral auxiliaries are also commonly used.[13,26-28]
The syntheses of 4 and 5 are illustrated in Scheme 2; complete
step-by-step synthetic details and structures may be found in
the Supporting Information. As with our previous syntheses of
stabilized PtdIns(3,4,5)P3 analogues,[13,14] we first prepared the
3,4,5-unprotected inositol using methods developed by
Bruzik:[27,28] 1) acetalization of inositol with d-camphor dimethyl
acetal, 2) selective silylation of 1-OH, 3) removal of the cam-
phor ketal, 4) selective benzoylation of 3,4,5-OH, 5) complete
protection of the hydroxyl groups, and 6) debenzoylation.
Next, the appropriately protected 5-PT intermediate 10, which
bears a free 1-hydroxyl group for introduction of the diacylgly-
ceryl-bearing phosphodiester, was prepared in 14 steps after
1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (TIPDS)-bis-silyla-
tion, benzoylation, desilylation, and phosphorylation, as depict-
ed in Scheme 2 (see the Supporting Information for complete
experimental details). In the same way, the appropriately pro-
tected 5-MP intermediate 15, equipped with a free 1-hydroxyl
group, was synthesized from myo-inositol in 11 steps
(Scheme 2; see the Supporting Information for complete ex-
perimental details) in close analogy to the synthesis[13] of 3-MP-
PtdIns(3,4,5)P3 (2).
Herein, we report the total asymmetric synthesis and charac-
terization of the two 5-stabilized PtdIns(3,4,5)P3 analogues 4
and 5 (Scheme 1). First, we investigated the relative binding af-
finities of the 3-, 5-, and 3,4,5-stabilized PtdIns(3,4,5)P3 ana-
logues to Grp1 PH domain using NMR titration methods.
Second, we examined the relative efficiency of inhibition of
SHIP1, SHIP2, and PTEN by these analogues, often with unex-
pected results. The reduced processing of the analogues as
substrates in an enzyme and modification-selective fashion
was also studied. Third, we explored the effects of the exoge-
nously delivered 3-, 5-, and 3,4,5-stabilized PtdIns(3,4,5)P3 ana-
logues on complement factor C5a-mediated polarization and
migration of wild-type murine neutrophils. Finally, the long-
lived agonist activities of the 5-PT (4) and 5-MP (5) analogues
in mimicking insulin action in sodium transport in A6 cells
were compared to previous data for the 3-PT (1), 3-MP (2), and
3,4,5-PT3 (3) analogues.[13,14]
Results and Discussion
Chemistry and synthesis
The PTEN resistant analogues, 3-PT-PtdIns(3,4,5)P3 (1),[13] 3-MP-
PtdIns(3,4,5)P3 (2),[13] and 3,4,5-PT-PtdIns(3,4,5)P3 (3)[14] have
ChemBioChem 2010, 11, 388 – 395
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
389

G. D. Prestwich et al.
Scheme 2. Synthesis of 5-PT and 5-MP analogues. a) BzCl, 4-dimethylamino pyridine, Py; b) tetrabutyl ammonium fluoride (TBAF), THF; c) dimethyl N,N-diiso-
propyl-phosphoramidite, 1H-tetrazole, meta-chloroperbenzoic acid; d) TBAF, DMF; e) triethylchlorosilane, imidazole; f) diisobutylaluminum hydride, CH2Cl2;
g) (iPr)2NP(OCH2CH2CN)2, 1H-tetrazole, phenylacetyl disulfide; h) NH4F, MeOH; i) 1H-tetrazole, CH2Cl2, RT, tBuOOH; j) triethylamine, BSTFA, CH3CN; k) trimethyl-
silyl bromide/CH2Cl2 (2:3), RT; l) MeOH; m) (MeO)2POCH2OTf, nBuLi, hexamethyl phosphoramide; n) 11, 1H-tetrazole, CH2Cl2, RT, tBuOOH. CE=cyanoethyl;
MOM=methoxymethyl ether; R=C3H7, C7H15, C15H31, C17H33.
The phosphoroamidates 11 were prepared from 2-cyanoeth-
yl-N,N-diisopropylchlorophosphoramidite with diacylglycerols
bearing dibutanoyl, dioctanoyl, or dihexadecanoyl chains,[13,18]
and coupled with 5-PT intermediate 10 or with 5-MP inter-
mediate 15, in the presence of 1H-tetrazole. Phosphodiesters
were produced by t-BuOOH oxidation, and no overoxidation of
the 5-PT group was observed. The cyanoethyl groups were re-
moved by using triethylamine plus bis(trimethylsilyl)trifluoroa-
cetamide (BSTFA) in anhydrous acetonitrile.[13,14] Addition of
BSTFA prevents the phosphorothioate anion from undergoing
re-alkylation. Protocols (see Supporting Information) analogous
to those for the 3-MP and 3-PT-PtdIns(3,4,5)P3 analogue syn-
theses[13] provided the desired analogues 5-PT-PtdIns(3,4,5)P3
(4) and 5-MP-PtdIns(3,4,5)P3 (5)[13,14] in the acid form. The final
products 4 and 5 were converted to the sodium salts by ion-
exchange chromatography (Dowex 50@ꢁ8 200 Na+ exchange
resin) and stored lyophilized at ꢀ808C prior to use in biologi-
cal experiments.
High-affinity binding to Grp1 PH
In mammalian cells, PtdIns(3,4,5)P3 is transiently accumulated
in plasma membranes following activation of PI 3-kinase.[29]
PtdIns(3,4,5)P3 is specifically recognized by the pleckstrin ho-
mology (PH) domain-containing proteins such as general re-
ceptor for phosphoinositides 1 (GRP1).[30,31] To test the biologi-
cal activity of the PtdIns(3,4,5)P3 analogues, we investigated
their interactions with GRP1 PH domain by NMR spectroscopy
(Figure 1 and Figure S1). The GRP1 PH domain recognizes all
five PtdIns(3,4,5)P3 analogues. The chemical-shift changes ob-
served in 1H,15N HSQC spectra of the 15N-labeled GRP1 PH
domain indicate that the binding of 3-PT-PtdIns(3,4,5)P3 (1)
390
www.chembiochem.org
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2010, 11, 388 – 395

Phosphatidylinositol Trisphosphate Analogues
somewhat unexpected. One
would have expected that 5-sta-
bilization should not completely
abrogate 3-phosphate removal
from an unstabilized position.
An alternative explanation is
that the chemical modifications
in 5-position of the 5-ms ana-
logues are not well tolerated in
the enzyme active site, and
simply result in poor enzyme–
substrate binding rather than
binding with reduced turnover.
Competitive inhibition of
SHIP1, SHIP2 and PTEN
1
Figure 1. PtdIns(3,4,5)P3 analogues are recognized by the PH domain of GRP1. Superimposed H,15N HSQC spectra
of 0.2 mm 15N-labeled GRP1 PH domain collected before (black) and after (gray) addition of indicated diC4-PtdIns-
(3,4,5)P3 analogues. The chemical shift changes for the amide group of Ile360 are indicated by arrows for compari-
son.
We chose to work with
Ins(1,3,4,5)P4 as substrate in
order to use relatively low sub-
strate concentrations, taking ad-
and 5-PT-PtdIns(3,4,5)P3 (4) is nearly equivalent to the binding
of unmodified PtdIns(3,4,5)P3. Figure S1 shows an overlay of
1
the H,15N HSQC spectra of GRP1 PH collected as three dibuta-
noyl lipids were added (unmodified PtdIns(3,4,5)P3, 3-PT-PtdIns-
(3,4,5)P3 (1), and 3,4,5-PT3-PtdIns(3,4,5)P3 (3)). The slow ex-
change regime on the NMR timescale in all three experiments
suggests a robust lipid–protein interaction. Taken together,
these data indicate that the binding affinity decreases in the
order PtdIns(3,4,5)P3 ꢁ3-PT-PtdIns(3,4,5)P3 (1)ꢂ5-PT-PtdIns-
(3,4,5)P3 (4)>3-MP-PtdIns(3,4,5)P3 (2)ꢂ5-MP-PtdIns(3,4,5)P3
(5)>3,4,5-PT3-PtdIns(3,4,5)P3 (3). It is important to note that,
while the phosphorothioate is considered isosteric with the
phosphate, the methylene phosphonate is a methylene-ex-
tended analogue of the phosphate, rather than an isostere.
Thus, these synthetic analogues can effectively substitute for
PtdIns(3,4,5)P3 in biochemical and biological assays while re-
taining affinity for PtdIns(3,4,5)P3 binding sites.
Figure 2. Phosphate release from metabolically stabilized analogues by
SHIP2 in the presence of 200 mm PtdSer,[49] as detected by malachite green
assay. Analogues, including the positive control diC8-PtdIns(3,4,5)P3, were
added at 100 mm. The final enzyme concentrations were 3.4 mgmLꢀ1, and in-
cubation was for 10 min at 378C. Enzyme activity is expressed as pmol of
phosphate released (means of triplicatesꢃSD).
Stabilized analogues are poor substrates for SHIP2 and
PTEN
Incubation of 100 mm of diC8-5-MP (5), diC8-5-PT (4), or diC8-
3,4,5-PT3 (3) with SHIP2 for 10 min resulted in no phosphate
release, thus confirming the metabolic stability of these 5-sta-
bilized analogues to the 5-phosphatase activity (Figure 2). In
contrast, both the 3-MP (2) and 3-PT (1) substrates were at
least partially dephosphorylated by SHIP2; this shows that the
5-phosphatase activity was capable of removing an unstabi-
lized phosphomonoester in the analogues containing a 3-stabi-
lized group.
vantage of the available tritium-labeled tracer and a well-char-
acterized assay[32] (see Experimental Section). The alternative
malachite green phosphate-release assay used to examine the
substrate ability of the analogues required much higher sub-
strate concentrations (50–100 mm).
The new 5-stabilized PtdIns(3,4,5)P3 analogues as well as the
previously described 3-stabilized PtdIns(3,4,5)P3 analogues[13]
and 3,4,5-tris(phosphorothioate) analogue[14] of PtdIns(3,4,5)P3
were examined as potential inhibitors of the 5-phosphatase
and 3-phosphatase activities. Figure 4A summarizes the com-
parative inhibition of the 5-phosphatase activity of recombi-
nant SHIP1, with 0.3 mm tritium-labeled Ins(1,3,4,5)P4 as the
substrate and each of the diC8 analogues at 10 mm. DiC8-
In contrast, none of the five analogues was processed by
PTEN during the 10 min incubation period (Figure 3). The ab-
sence of phosphate release confirms the metabolic stability of
these analogues to the 3-phosphatase activity of PTEN, but is
ChemBioChem 2010, 11, 388 – 395
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
391

G. D. Prestwich et al.
Figure 3. Phosphate release from metabolically stabilized analogues by
PTEN in the presence of 200 mm PtdSer,[49] as detected by malachite green
assay. Analogues, including the positive control diC8-PtdIns(3,4,5)P3, were
added at 100 mm at 378C. The final enzyme concentration was
0.125 mgmLꢀ1 and incubation time was 10 min. Enzyme activity is as de-
scribed in the legend for Figure 2.
PtdIns(3,4,5)P3 itself did not show any competitive inhibition of
SHIP1 activity under these conditions, but each of the ana-
logues significantly reduced SHIP1 activity. Interestingly, the
most potent analogues in this assay were diC8-5-MP (5), diC8-3-
PT (1), and diC8-3,4,5-PT3 (3). In contrast to the situation with
SHIP1, little or no inhibition of SHIP2 activity was observed for
any of the analogues under the same incubation conditions
(Figure 4B). This most likely reflects the difference in affinity of
the substrate Ins(1,3,4,5)P4 for SHIP1 and SHIP2.[32]
Evaluation of the five stabilized analogues for inhibition of
PTEN 3-phosphatase activity is shown in Figure S2, for which
0.3 mm tritium-labeled Ins(1,3,4,5)P4 as the substrate and 10 mm
of each of the diC8 analogues were employed. Only the 3,4,5-
PT3 analogue (3) appeared to inhibit PTEN 3-phosphatase ac-
tivity with this substrate.
Figure 4. Inhibition of the 5-phosphatase activity of A) recombinant SHIP1 at
4.9 mgmLꢀ1 and B) recombinant SHIP2 at 11.3 mgmLꢀ1, with 0.3 mm tritium-
labeled Ins(1,3,4,5)P4 as the substrate and each of the diC8 analogues at
10 mm. Analogues were preincubated for 5 min in the presence of enzyme
at 48C. The mixture was then mixed with the labeled substrate and incubat-
ed for 10 min at 378C. The 100% control refers to the activity in the pres-
ence of water, while each value below 100% represents the activity that was
reached in the presence of analogue (mean valueꢃSD).
Neutrophil polarity and motility
Chemotaxis is a crucially important cellular response implicated
in physiological activities.[33-36] Chemotactic stimulation of cells
results in a complex sequence of events: increased actin or-
ganization, cell-shape changes, and the development of polari-
ty.[37,38] The selective translocation of PtdIns(3,4,5)P3 to the
plasma membrane at the leading edge leads to cytoskeleton
reorganization and pseudopod formation,[39] so the localized
accumulation of PtdIns(3,4,5)P3 was deemed to be the key
event directing the recruitment and activation of the signaling
components required for cell polarization and chemotaxis.[40,41]
The amoeba Dictyostelium discoideum and mammalian neutro-
phils are commonly used as representative chemotactic cells.
In D. discoideum, PI(3)Ks and PTEN control cell polarization and
chemotaxis by regulating spatially localized PtdIns(3,4,5)P3 ac-
cumulation.[42,43] In neutrophils, both PI3Kg and SHIP1 play criti-
cal roles in polarization and motility;[36,44] SHIP1 restricts the lo-
calization of PtdIns(3,4,5)P3 accumulation, and thereby governs
the cell polarization required for proper motility and chemo-
taxis.[36] Thus, we examined the effects of metabolically stabi-
lized PtdIns(3,4,5)P3 analogues in wild-type neutrophils by
using C5a as chemoattractant, which could in principle occur
by a possible interaction with SHIP1 or with Akt or both. As
shown in Figure 5, the polarization of wild-type neutrophils
was greatly impaired by exogenous addition of each of the
five PtdIns(3,4,5)P3 analogues. The 3- and 5-ms-PtdIns(3,4,5)P3
analogues might bind the SHIP1 as an inhibitor or antagonist,
thus the ability of SHIP1 to govern PtdIns(3,4,5)P3 and/or
PtdIns(3,4)P2 accumulation at the leading edge of chemotaxing
neutrophils is decreased, the formation of the pseudopod is re-
tarded.
To determine if the metabolically stabilized analogues might
be acting at the level of Akt activation, mouse peritoneal neu-
trophils were purified, stimulated, and lysed as described.[36]
The lysates were separated by 10% SDS-PAGE and immuno-
blotted with phospho-specific antibodies against Akt pS473
and pan-Akt. As shown in Figure S3, none of the five ana-
392
www.chembiochem.org
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2010, 11, 388 – 395

Phosphatidylinositol Trisphosphate Analogues
PtdIns(3,4,5)P3 (5) on sodium transport across conflu-
ent monolayers of A6 cells. As shown in Figure S4,
apical addition of analogues 4 and 5 increased
sodium transport. The 5-ms analogues 4 and 5 also
mimicked the activity of unstabilized PtdIns(3,4,5)P3.
It is worth noting that there was no significant varia-
tion in the resistance of the monolayers of A6 cells
upon addition of the PtdIns(3,4,5)P3 analogues tested
in this study up to 60 min. Moreover, addition of ami-
loride to the apical bathing medium completely in-
hibited the current stimulated by either insulin or
PtdIns(3,4,5)P3.[46] We propose that these analogues
might have other applications as pharmacological
tools to probe role of PtdIns(3,4,5)P3 in a cellular con-
text.
Conclusions
Two 5-phosphatase-resistant analogues of PtdIns-
(3,4,5)P3 were synthesized and characterized in bio-
chemical and biological contexts. First, the modifica-
tion of a single phosphate of PtdIns(3,4,5)P3 resulted
in retention of binding to the PtdIns(3,4,5)P3-specific
GRP1 PH domain, with varying relative affinities.
Second, modest inhibition of the action of SHIP1
phosphatase activity on Ins(1,3,4,5)P4 was evident,
and less marked inhibition of SHIP2 or PTEN dephos-
phorylation of Ins(1,3,4,5)P4 by any of the analogues
was observed. Third, while none of the five phospha-
tase-resistant analogues released phosphate when in-
cubated with the 3-phosphatase PTEN, the 3-stabi-
lized analogues 1 and 2 were partially dephosphory-
lated by the 5-phosphatase activity. Fourth, each of
the analogues severely impaired complement factor C5a-medi-
ated polarization and migration of murine neutrophils, with 3-
MP (2) showing the greatest effect on polarization and 3,4,5-
PT3 (3) leading to the greatest decrease in eccentricity. Finally,
the new 5-stabilized analogues 4 and 5, similar to the previ-
ously tested analogues 1, 2, and 3, both activated sodium
transport in A6 cells.
Figure 5. Effects of metabolically stabilized PtdIns(3,4,5)P3 analogues on cell polarization.
Mouse neutrophils (4ꢁ105) were preincubated with or without metabolically stabilized
PtdIns(3,4,5)P3, followed by the addition of C5a and a further incubation for 15 min. Sub-
sequently, cells were stained with fluorescent phalloidin, and the percentage of A) polar-
ized cells, B) roundness, or C) eccentricity was determined as described in the Experimen-
tal Section. Higher values of roundness and eccentricity indicate that the cell shape is
further from a circle and that the cell is more polarized. All data are presented as the
meanꢃSD. Significant differences in each parameter from cells treated with C5a in the
absence of a PtdIns(3,4,5)P3 analogue are indicated by asterisks: *: p<0.05, **: p<0.01
versus control (histone H1 carrier) (Student t-test). D) Selected micrographs of inactivated
(top) or C5a-activiated (bottom) neutrophils treated with no ligand or one of the two
most potent ligands, 5-PT-PtdIns(3,4,5)P3 or 3,4,5-PT3-PtdIns(3,4,5)P3.
logues activated Akt phosphorylation, and the two analogues
tested, 3-PT and 3,4,5-PT3, failed to inhibit C5a-stimulated Akt
phosphorylation.
We had hypothesized that the 5-stabilized analogues would
be able to prevent the physiological production of PtdIns-
(3,4)P2 that is, at least in part, produced by the action of SHIP1
on PtdIns(3,4,5)P3. In other words, by interfering with SHIP1 ac-
tivity, we had envisaged that the exogenous addition of an an-
alogue such as diC8-3,4,5-PT3-PtdIns(3,4,5)P3 could qualitatively
phenocopy the effect of the absence of SHIP1 in altering neu-
trophil polarization[36] (see Figure 5). In the end, however, this
desired result was not achieved.
We had originally envisaged using these new analogues as
phosphatase-resistant mimics of PtdIns(3,4,5)P3 to selectively
manipulate cell responses. Unfortunately, the complexity of dif-
ferential interactions of the analogues with different PtdIns-
(3,4,5)P3 targets, for example, Akt, GRP1, and PDK1, rendered
this expectation unrealistic in a cellular context.
Sodium transport
Experimental Section
To test the function of these analogues, we used A6 cell mono-
layers, a renal epithelium model that expresses epithelial
sodium channels (ENaC), in which carrier-mediated intracellular
delivery[45] of PtdIns(3,4,5)P3 activates sodium transport.[46]
ENaC activity is the rate-limiting step of the sodium transport
and is stimulated by insulin.[47] DiC16-PtdIns(3,4,5)P3 is an early
mediator of the insulin-stimulated sodium transport in A6 cells.
Thus, we compared the effect of the unmodified diC16-PtdIns-
(3,4,5)P3 with diC16-5-PT-PtdIns(3,4,5)P3 (4) and diC16-5-MP-
Chemical syntheses: The synthesis of the 3-metabolically stabi-
lized PtdIns(3,4,5)P3 analogues 1, 2 and 3,4,5-PT-PtdIns(3,4,5)P3 (3)
have been previously published. The 5-stabilized PtdIns(3,4,5)P3 an-
alogues 4 and 5 were synthesized in a similar way, and full details
for their preparation and characterization can be found in the Sup-
porting Information.
Protein expression and purification: The DNA fragment encoding
residues 261–385 of the PH domain of human GRP1 was cloned in
ChemBioChem 2010, 11, 388 – 395
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
393

G. D. Prestwich et al.
pRSET A vector (Invitrogen). The 15N-labeled protein was expressed
in E. coli Rosetta in minimal medium supplemented with 15NH4Cl
(Cambridge Isotope). Bacteria were harvested by centrifugation
after induction with isopropyl-b-d-thiogalactopyranoside (IPTG,
0.1 mm) and lysed in a French Press. The His6 fusion protein was
purified on a Talon resin column (Clontech Laboratories, Inc.). The
His tag was cleaved with EKMax (Invitrogen). The protein was fur-
ther purified by ion-exchange chromatography on a HiTrap SP HP
column (Amersham) in Bis-Tris buffer (pH 6.5), and concentrated in
Millipore concentrators.
Experimental determination and evaluation of sodium transport
(INaþ [mAcmꢀ2]): Briefly, A6 cells were subcultured onto 24 mm Mil-
licell inserts (Millipore) for 10 days and, the day before the experi-
ment, incubated overnight in a serum-free 260 mosmol per kg H2O
amphibian Ringer solution. DiC16-PtdIns(3,4,5)P3, analogues 4 and 5
was complexed by histone H1 carrier (50 mm) and then added to
the apical side of the monolayer. The results were compared with
insulin basolateral stimulation (100 nm) and control (histone H1
alone). This experiment is representative of three independent ex-
periments. (See Figure S4.)
NMR spectroscopy and titration of PtdIns(3,4,5)P3 analogues:
NMR spectra were recorded at 258C on a Varian INOVA 500 MHz
Acknowledgements
1
spectrometer. The H,15N HSQC spectra of 0.2 mm uniformly 15N-la-
beled PH domain were collected while dibutanoyl (C4)-PtdIns-
(3,4,5)P3 analogues were added stepwise. (See also Figure S1).
We thank the NIH (NS 29632 to G.D.P. and GM 071424 to T.G.K.).
The research of T.S. was supported by a Grant-in-Aid for Creative
Scientific Research and a Grant for Young Scientists (A) from JSPS
and MEXT. C.E. was supported by a grant from the Interuniversity
Attraction Poles Programme (P6/28)—Belgium State—Belgium
Science Policy and by the Fonds de la Recherche Scientifique
Mꢀdicale. We thank Colette Moreau for her help in conducting
the inositol phosphatase assays.
Expression of human SHIP1 and SHIP2 enzymes. The SHIP2 cata-
lytic domain was expressed as a glutathione-S-transferase (GST)
fusion protein and expressed in bacteria as reported.[49] The cDNA
encoding human SHIP1 was subcloned in pTrHis bacterial expres-
sion vector (this construct was provided by, Dr Len Stephens, Bab-
raham, UK) and expressed as His-tagged fusion protein.[50] Briefly,
an overnight culture of bacteria (0.4 L) was twofold diluted the
next day to reach an OD600 of 0.6 and induced by IPTG for 5 h at
208C. The crude lysate was taken up in Tris-HCl (50 mm, pH 7.5)
containing 0.1% Triton X-100, NaCl (300 mm), 10% glycerol, MgCl2
(5 mm), and complete protease inhibitors (Roche). The enzyme was
applied to a Ni-NTA agarose column (Qiagen), washed, and eluted
with imidazole (100 mm) in the same buffer. GST-PTEN was ex-
pressed and purified as reported.[49]
Keywords: asymmetric synthesis · enzyme inhibition · inositol
lipid · metabolic stability · phosphatases · sodium transport
[1] G. D. Prestwich, Chem. Biol. 2004, 11, 619–637.
[2] B. E. Drees, G. B. Mills, C. Rommel, G. D. Prestwich, Expert Opin. Ther. Pat.
2004, 14, 703–732.
[3] A. E. Traynor-Kaplan, A. L. Harris, B. L. Thompson, P. Taylor, L. A. Sklar,
Nature 1988, 334, 353–356.
[4] A. Toker, L. C. Cantley, Nature 1997, 387, 673–676.
[5] L. C. Cantley, K. R. Auger, C. Carpenter, B. Duckworth, A. Graziani, R. Ka-
peller, S. Soltoff, Cell 1991, 64, 281–302.
[6] N. Pinal, D. C. Goberdhan, L. Collinson, Y. Fujita, I. M. Cox, C. Wilson, F.
Pichaud, Curr. Biol. 2006, 16, 140–149.
[7] G. Sweeney, R. R. Garg, R. B. Ceddia, D. Li, M. Ishiki, R. Somwar, L. J.
Foster, P. O. Neilsen, G. D. Prestwich, A. Rudich, A. Klip, J. Biol. Chem.
2004, 279, 32233–32242.
[8] T. Maehama, J. E. Dixon, Trends Cell. Biol. 1999, 9, 125–128.
[9] X. Pesesse, S. Deleu, F. De Smedt, L. Drayer, C. Erneux, Biochem. Biophys.
Res. Commun. 1997, 239, 697–700.
[10] S. Harris, R. V. Parry, J. Westwick, S. G. Ward, J. Biol. Chem. 2008, 283,
2465–2469.
[11] D. Lazar, A. Saltiel, Nat. Rev. Drug Discovery 2006, 5, 333–342.
[12] D. Blero, B. Payrastre, S. Schurmans, C. Erneux, Pfluegers Arch. 2007, 455,
31–44.
Determination of SHIP1, SHIP2, and PTEN phosphatase activi-
ties: SHIP1 and SHIP2 phosphatase activities were determined in
the presence of [3H]-Ins(1,3,4,5)P4 (0.3 mm), and Dowex columns
was used to isolate the dephosphorylated product.[51] When the
substrate specificity of SHIP2 and PTEN was addressed with the
various analogues, the malachite phosphate release assay was
used as described.[49] The enzyme activity data are shown as means
of triplicatesꢃstandard deviation. (See Figure S2 for PTEN inhibi-
tion.)
Western blot for Akt activation: Mouse peritoneal neutrophils
were purified, stimulated, and lysed as described.[36] Lysates were
separated by 10% SDS-PAGE, followed by immunoblotting with
phospho-specific antibodies against Akt pS473 and pan-Akt (Cell
Signaling Technology). (See Figure S3).
Neutrophil morphology: Mouse neutrophils (4ꢁ105) were seeded
onto glass coverslips. Cells were allowed to attach for 10 min at
room temperature, incubated at 378C with or without diC16-PtdIns-
(3,4,5)P3 analogues (10 mm) and carrier (1.5 mm histone H1) for
30 min, and stimulated with complement factor 5a (C5a, 5 nm).
The reaction was stopped by fixing the cells in 4% paraformalde-
hyde. The fixed cells were washed and incubated for 1 h in PBS
[13] H. Zhang, N. Markadieu, R. Beauwens, C. Erneux, G. D. Prestwich, J. Am.
Chem. Soc. 2006, 128, 16464–16465.
[14] H. Zhang, Y. Xu, N. Markadieu, R. Beauwens, C. Erneux, G. D. Prestwich,
Bioorg. Med. Chem. Lett. 2008, 18, 762–766.
[15] D. Lampe, C. Liu, B. V. Potter, J. Med. Chem. 1994, 37, 907–912.
[16] A. W. Murray, M. R. Atkinson, Biochemistry 1968, 7, 4023–4029.
[17] A. Hampton, L. W. Brox, M. Bayer, Biochemistry 1969, 8, 2303–2311.
[18] Y. Xu, S. A. Lee, T. G. Kutateladze, D. Sbrissa, A. Shisheva, G. D. Prestwich,
J. Am. Chem. Soc. 2006, 128, 885–897.
containing Alexa Fluor 488 phalloidin (0.2 UmLꢀ1
; Molecular
[19] W. Huang, H. Zhang, F. Davrazou, T. G. Kutateladze, X. Shi, O. Gozani,
G. D. Prestwich, J. Am. Chem. Soc. 2007, 129, 6498–6506.
[20] L. Zhang, W. Huang, A. Tanimura, T. Morita, S. Harihar, D. B. Dewald,
G. D. Prestwich, ChemMedChem 2007, 2, 1281–1289.
[21] G. D. Prestwich, Acc. Chem. Res. 1996, 29, 503–513.
[22] K. K. Reddy, M. Saddy, J. R. Falck, J. Org. Chem. 1995, 60, 3385–3390.
[23] B. R. Sculimbrene, Y. Xu, S. J. Miller, J. Am. Chem. Soc. 2004, 126, 13182–
13183.
Probes), 0.1% lysophosphatidylcholine (Nacalai, Kyoto, Japan), and
1% BSA. Cell shape was determined from confocal images of fluo-
rescently labeled cells. Cells with distinctive, F-actin-rich leading
edges were qualified as polarized (n>300 cells). The perimeter (x),
area (y), eccentricity, and radial standard deviation (radial S.D.)
were determined for each cell (n>43) by using the analysis func-
tions of IPLab software (Scanalytics, Fairfax, VA). The formula used
to determine roundness (R) was R=1ꢀ4pyxꢀ2. Low values for ec-
centricity, radial S.D. and roundness indicate a lack of polarization.
[24] B. R. Sculimbrene, S. J. Miller, J. Am. Chem. Soc. 2001, 123, 10125–
10126.
394
www.chembiochem.org
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2010, 11, 388 – 395

Phosphatidylinositol Trisphosphate Analogues
[25] D.-S. Wang, C.-S. Chen, J. Org. Chem. 1996, 61, 5905–5910.
[26] H. Zhang, Y. Xu, Z. Zhang, E. R. Liman, G. D. Prestwich, J. Am. Chem. Soc.
2006, 128, 5642–5643.
[40] S. G. Ward, Trends Immunol. 2004, 25, 67–74.
[41] M. P. Wymann, S. Sozzani, F. Altruda, A. Mantovani, E. Hirsch, Immunol.
Today 2000, 21, 260–264.
[27] K. S. Bruzik, M.-D. Tsai, J. Am. Chem. Soc. 1992, 114, 6361–6374.
[28] R. J. Kubiak, K. S. Bruzik, J. Org. Chem. 2003, 68, 960–968.
[29] B. Vanhaesebroeck, S. J. Leevers, K. Ahmadi, J. Timms, R. Katso, P. C. Dris-
coll, R. Woscholski, P. J. Parker, M. D. Waterfield, Annu. Rev. Biochem.
2001, 70, 535–602.
[30] K. M. Ferguson, J. M. Kavran, V. G. Sankaran, E. Fournier, S. J. Isakoff, E. Y.
Skolnik, M. A. Lemmon, Mol. Cell. 2000, 6, 373–384.
[31] S. E. Lietzke, S. Bose, T. Cronin, J. Klarlund, A. Chawla, M. P. Czech, D. G.
Lambright, Mol. Cell 2000, 6, 385–394.
[32] X. Pesesse, C. Moreau, A. L. Drayer, R. Woscholski, P. Parker, C. Erneux,
FEBS Lett. 1998, 437, 301–303.
[33] V. Niggli, Int. J. Biochem. Cell. Biol. 2003, 35, 1619–1638.
[34] H. T. Park, J. Wu, Y. Rao, Bioessays 2002, 24, 821–827.
[35] P. Rickert, O. D. Weiner, F. Wang, H. R. Bourne, G. Servant, Trends. Cell.
Biol. 2000, 10, 466–473.
[36] M. Nishio, K.-i. Watanabe, J. Sasaki, C. Taya, S. Takasuga, R. Iizuka, T.
Balla, M. Yamazaki, H. Watanabe, R. Itoh, S. Kuroda, Y. Horie, I. Fçrster,
T. W. Mak, H. Yonekawa, J. M. Penninger, Y. Kanaho, A. Suzuki, T. Sasaki,
Nat. Cell. Biol. 2007, 9, 36–44.
[37] A. J. Ridley, M. A. Schwartz, K. Burridge, R. A. Firtel, M. H. Ginsberg, G.
Borisy, J. T. Parsons, A. R. Horwitz, Science 2003, 302, 1704–1709.
[38] L. Stephens, C. Ellson, P. Hawkins, Curr. Opin. Cell. Biol. 2002, 14, 203–
213.
[42] M. Iijima, P. Devreotes, Cell 2002, 109, 599–610.
[43] S. Funamoto, R. Meili, S. Lee, L. Parry, R. A. Firtel, Cell 2002, 109, 611–
623.
[44] T. Sasaki, J. Irie-Sasaki, R. Jones, A. Oliveira-dos-Santos, W. Stanford, B.
Bolon, A. Wakeham, A. Itie, D. Bouchard, I. Kozieradzki, N. Joza, T. Mak,
P. Ohashi, A. Suzuki, J. M. Penninger, Science 2000, 287, 1040–1046.
[45] S. Ozaki, D. B. DeWald, J. C. Shope, J. Chen, G. D. Prestwich, Proc. Natl.
Acad. Sci. USA 2000, 97, 11286–11291.
[46] N. Markadieu, D. Blero, A. Boom, C. Erneux, R. Beauwens, Am. J. Physiol.
Renal. Physiol. 2004, 287, F319–F328.
[47] B. C. Rossier, C. M. Canessa, L. Schild, J. D. Horisberger, Curr. Opin. Neph-
rol. Hypertens. 1994, 3, 487–496.
[48] Y. Jia, K. Subramanian, C. Erneux, V. Pouillon, H. Hattori, H. Jo, J. You, D.
Zhu, S. Schurmans, H. Luo, Immunity 2007, 27, 453–467.
[49] F. Vandeput, K. Backers, V. Villeret, X. Pesesse, C. Erneux, Cell Signal.
2006, 18, 2193–2199.
[50] A. L. Drayer, X. Pesesse, F. De Smedt, R. Woscholski, P. J. Parker, C.
Erneux, Biochem. Biophys. Res. Commun. 1996, 225, 243–249.
[51] C. Erneux, M. Lemos, G. Verjans, P. Vanderhaeghen, Delvaux, A. J.
Dumont, Eur. J. Biochem. 1989, 181, 317–322.
Received: August 30, 2009
[39] G. Servant, O. D. Weiner, P. Herzmark, T. Balla, J. W. Sedat, H. R. Bourne,
Science 2000, 287, 1037–1040.
Published online on January 5, 2010
ChemBioChem 2010, 11, 388 – 395
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
395